Growth differentiation factor-8 family nucleic acid sequences

ABSTRACT

Growth differentiation factor-8 (GDF-8) is disclosed along with its polynucleotide sequence and amino acid sequence. Also disclosed are diagnostic and therapeutic methods of using the GDF-8 polypeptide and polynucleotide sequences.

This application is a continuation and claims the benefit of priorityunder 35 USC §120 of U.S. application Ser. No. 09/177,860, filed Oct.23, 1998, issued Aug. 1, 2000, U.S. Pat. No. 6,095,506; which is adivisional of U.S. application Ser. No. 08/525,596 filed Oct. 26, 1995,issued Oct. 27, 1998 as U.S. Pat. No. 5,827,733; which is a §371application of PCT US94/03019, filed Mar. 18, 1994; which is acontinuation-in-part of U.S. application Ser. No. 08/033,923, filed Mar.19, 1993 (abandoned). The disclosures of the prior applications areconsidered part of and are incorporated by reference in the disclosureof this application.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates generally to growth factors and specifically to anew member of the transforming growth factor beta (TGF-β) superfamily,which is denoted, growth differentiation factor-8 (GDF-8).

2. Description of Related Art

The transforming growth factor β (TGF-β) superfamily encompasses a groupof structurally-related proteins which affect a wide range ofdifferentiation processes during embryonic development. The familyincludes, Mullerian inhibiting substance (MIS), which is required fornormal male sex development (Behringer, et al., Nature, 345:167, 1990),Drosophila decapentaplegic (DPP) gene product, which is required fordorsal-ventral axis formation and morphogenesis of the imaginal disks(Padgett, et al., Nature, 325:81-84, 1987), the Xenopus Vg-1 geneproduct, which localizes to the vegetal pole of eggs ((Weeks, et al.,Cell, 51:861-867, 1987), the activins (Mason, et al., Biochem, Biophys.Res. Commun., 135:957-964, 1986), which can induce the formation ofmesoderm and anterior structures in Xenopus embryos (Thomsen, et al.,Cell, 63:485, 1990), and the bone morphogenetic proteins (BMPs,osteogenin, OP-1) which can induce de novo cartilage and bone formation(Sampath, et al., J. Biol. Chem., 265:13198, 1990). The TGF-βs caninfluence a variety of differentiation processes, includingadipogenesis, myogenesis, chondrogenesis, hematopoiesis, and epithelialcell differentiation (for review, see Massague, Cell 49:437, 1987).

The proteins of the TGF-β family are initially synthesized as a largeprecursor protein which subsequently undergoes proteolytic cleavage at acluster of basic residues approximately 110-140 amino acids from theC-terminus. The C-terminal regions, or mature regions, of the proteinsare all structurally related and the different family members can beclassified into distinct subgroups based on the extent of theirhomology. Although the homologies within particular subgroups range from70% to 90% amino acid sequence identity, the homologies betweensubgroups are significantly lower, generally ranging from only 20% to50%. In each case, the active species appears to be a disulfide-linkeddimer of C-terminal fragments. Studies have shown that when thepro-region of a member of the TGF-β family is coexpressed with a matureregion of another member of the TGF-β family, intracellular dimerizationand secretion of biologically active homodimers occur (Gray, A., andMaston, A., Science, 247:1328, 1990). Additional studies by Hammonds, etal., (Molec. Endocrin. 5:149, 1991) showed that the use of the BMP-2pro-region combined with the BMP-4 mature region led to dramaticallyimproved expression of mature BMP-4. For most of the family members thathave been studied, the homodimeric species has been found to bebiologically active, but for other family members, like the inhibins(Ling, et al., Nature, 321:779, 1986) and the TGF-βs (Cheifetz, et al.,Cell, 48:409, 1987), heterodimers have also been detected, and theseappear to have different biological properties than the respectivehomodimers.

Identification of new factors that are tissue-specific in theirexpression pattern will provide a greater understanding of that tissue'sdevelopment and function.

SUMMARY OF THE INVENTION

The present invention provides a cell growth and differentiation factor,GDF-8, a polynucleotide sequence which encodes the factor, andantibodies which are immunoreactive with the factor. This factor appearsto relate to various cell proliferative disorders, especially thoseinvolving those involving muscle, nerve, and adipose tissue.

Thus, in one embodiment, the invention provides a method for detecting acell proliferative disorder of muscle, nerve, or fat origin and which isassociated with GDF-8. In another embodiment, the invention provides amethod for treating a cell proliferative disorder by suppressing orenhancing GDF-8 activity.

DESCRIPTION OF THE DRAWINGS

FIG. 1 is a Northern blot showing expression of GDF-8 mRNA in adulttissues. The probe was a partial murine GDF-8 clone.

FIG. 2 shows nucleotide and predicted amino acid sequences of murineGDF-8 (FIG. 2a; SEQ ID NO: 12) and human GDF-8 (FIG. 2b; SEQ ID NO: 14).The putative dibasic processing sites in the murine sequence are boxed.

FIG. 3 shows the alignment of the C-terminal sequences of GDF-8 (see,for example, amino acids 265 to 376 of SEQ ID NO: 12) with other membersof the TGF-β superfamily (SEQ ID NOS: 18 to 31). The conserved cysteineresidues are boxed. Dashes denote gaps introduced in order to maximizealignment.

FIG. 4 shows amino acid homologies among different members of the TGF-βsuperfamily. Numbers represent percent amino acid identities betweeneach pair calculated from the first conserved cysteine to theC-terminus. Boxes represent homologies among highly-related memberswithin particular subgroups.

FIG. 5 shows nucleotide and deduced amino acid sequences of GDF-8. Amurine GDF-8 nucleotide sequence (SEQ ID NO: 11) and deduced amino acidsequence (SEQ ID NO: 12; FIGS. 5a and 5 b) and a human GDF-8 nucleotidesequence (SEQ ID NO: 13) and deduced amino acid sequence (SEQ ID NO: 14;FIGS. 5c and 5 d) are shown. Numbers indicate nucleotide positionrelative to the 5′ end. Consensus N-linked glycosylation signals areshaded. The putative RXXR (SEQ ID NO: 32) proteolytic cleavage sites areboxed.

FIG. 6 shows a hydropathicity profile of GDF-8. Average hydrophobicityvalues for murine (FIG. 6a) and human (FIG. 6b) GDF-8 were calculatedusing the method of Kyte and Doolittle (J. Mol. Biol., 157:105-132,1982). Positive numbers indicate increasing hydrophobicity.

FIG. 7 shows a comparison of murine and human GDF-8 amino acidsequences. The predicted murine sequence (SEQ ID NOS: 12 and 14,respectively), is shown in the top lines and the predicted humansequence is shown in the bottom lines. Numbers indicate amino acidposition relative to the N-terminus. Identities between the twosequences are denoted by a vertical line.

FIG. 8 shows the expression of GDF-8 in bacteria. BL21 (DE3) (pLysS)cells carrying a pRSET/GDF-8 expression plasmid were induced withisopropylthio-β-galactoside, and the GDF-8 fusion protein was purifiedby metal chelate chromatography. Lanes: total=total cell lysate;soluble=soluble protein fraction; insoluble=insoluble protein fraction(resuspended in 10 mM Tris pH 8.0, 50 mM sodium phosphate, 8 M urea, and10 mM β-mercaptoethanol [buffer B]) loaded onto the column;pellet=insoluble protein fraction discarded before loading the column;flowthrough=proteins not bound by the column; washes=washes carried outin buffer B at the indicated pH's. Positions of molecular weightstandards are shown at the right. Arrow indicates the position of theGDF-8 fusion protein.

FIG. 9 shows the expression of GDF-8 in mammalian cells. Chinese hamsterovary cells were transfected with pMSXND/GDF-8 expression plasmids andselected in G418. Conditioned media from G418-resistant cells (preparedfrom cells transfected with constructs in which GDF-8 was cloned ineither the antisense or sense orientation) were concentrated,electrophoresed under reducing conditions, blotted, and probed withanti-GDF-8 antibodies and [¹²⁵l]iodoproteinA. Arrow indicates theposition of the processed GDF-8 protein.

FIG. 10 shows the expression of GDF-8 mRNA. Poly A-selected RNA (5 μgeach) prepared from adult tissues (FIG. 10a) or placentas and embryos(FIG. 10b) at the indicated days of gestation was electrophoresed onformaldehyde gels, blotted, and probed with full length murine GDF-8.

FIG. 11 shows chromosomal mapping of human GDF-8. DNA samples preparedfrom human/rodent somatic cell hybrid lines were subjected to PCR,electrophoresed on agarose gels, blotted, and probed. The humanchromosome contained in each of the hybrid cell lines is identified atthe top of each of the first 24 lanes (1-22, X, and Y). In the lanesdesignated M, CHO, and H. the starting DNA template was total genomicDNA from mouse, hamster, and human sources, respectively. In the lanemarked B1, no template DNA was used. Numbers at left indicate themobilities of DNA standards.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a growth and differentiation factor,GDF-8 and a polynucleotide sequence encoding GDF-8. GDF-8 is expressedat highest levels in muscle and at lower levels in adipose tissue. Inone embodiment, the invention provides a method for detection of a cellproliferative disorder of muscle, nerve, or fat origin which isassociated with GDF-8 expression. In another embodiment, the inventionprovides a method for treating a cell proliferative disorder by using anagent which suppresses or enhances GDF8 activity.

The TGF-β superfamily consists of multifunctional polypeptides thatcontrol proliferation, differentiation, and other functions in many celltypes. Many of the peptides have regulatory, both positive and negative,effects on other peptide growth factors. The structural homology betweenthe GDF-8 protein of this invention and the members of the TGF-β family,indicates that GDF-8 is a new member of the family of growth anddifferentiation factors. Based on the known activities of many of theother members, it can be expected that GDF-8 will also possessbiological activities that will make it useful as a diagnostic andtherapeutic reagent.

In particular, certain members of this superfamily have expressionpatterns or possess activities that relate to the function of thenervous system. For example, the inhibins and activins have been shownto be expressed in the brain (Meunier, et al., Proc. Natl. Acad. Sci.,USA, 85:247, 1988; Sawchenko, et al., Nature, 334:615, 1988), andactivin has been shown to be capable of functioning as a nerve cellsurvival molecule (Schubert, et al., Nature, 344:868, 1990). Anotherfamily member, namely, GDF-1, is nervous system-specific in itsexpression pattern (Lee, S. J., Proc. Natl. Acad. Sci., USA, 88:4250,1991), and certain other family members, such as Vgr-1 (Lyons, et al.,Proc. Natl Acad. Sci., USA, 86:4554, 1989; Jones, et al., Development,111:531, 1991), OP-1 (Ozkaynak, et al., J. Biol. Chem., 267:25220,1992), and BMP-4 (Jones, et al., Development, 111:531, 1991), are alsoknown to be expressed in the nervous system. Because it is known thatskeletal muscle produces a factor or factors that promote the survivalof motor neurons (Brown, Trends Neurosci., 7:10, 1984), the expressionof GDF-8 in muscle suggests that one activity of GDF-8 may be as atrophic factor for neurons. In this regard, GDF-8 may have applicationsin the treatment of neurodegenerative diseases, such as amyotrophiclateral sclerosis, or in maintaining cells or tissues in culture priorto transplantation.

GDF-8 may also have applications in treating disease processes involvingmuscle, such as in musculodegenerative diseases or in tissue repair dueto trauma. In this regard, many other members of the TGF-β family arealso important mediators of tissue repair. TGF-β has been shown to havemarked effects on the formation of collagen and to cause a strikingangiogenic response in the newborn mouse (Roberts, et al., Proc. Natl.Acad. Sci., USA 83:4167, 1986). TGF-β has also been shown to inhibit thedifferentiation of myoblasts in culture (Massague, et al., Proc. Natl.Acad. Sci., USA 83:8206, 1986). Moreover, because myoblast cells may beused as a vehicle for delivering genes to muscle for gene therapy, theproperties of GDF-8 could be exploited for maintaining cells prior totransplantation or for enhancing the efficiency of the fusion process.

The expression of GDF-8 in adipose tissue also raises the possibility ofapplications for GDF-8 in the treatment of obesity or of disordersrelated to abnormal proliferation of adipocytes. In this regard, TGF-βhas been shown to be a potent inhibitor of adipocyte differentiation invitro (Ignotz and Massague, Proc. Natl. Acad. Sci., USA 82:8530, 1985).

The term “substantially pure” as used herein refers to GDF-8 which issubstantially free of other proteins, lipids, carbohydrates or othermaterials with which it is naturally associated. One skilled in the artcan purify GDF-8 using standard techniques for protein purification. Thesubstantially pure polypetide will yield a single major band on anon-reducing polyacrylamide gel. The purity of the GDF-8 polypeptide canalso be determined by amino-terminal amino acid sequence analysis. GDF-8polypeptide includes functional fragments of the polypeptide, as long asthe activity of GDF-8 remains. Smaller peptides containing thebiological activity of GDF-8 are included in the invention.

The invention provides polynucleotides encoding the GDF-8 protein. Thesepolynucleotides include DNA. CDNA and RNA sequences which encode GDF-8.It is understood that all polynucleotides encoding all or a portion ofGDF-8 are also included herein, as long as they encode a polypeptidewith GDF-8 activity. Such polynucleotides include naturally occurring,synthetic, and intentionally manipulated polynucleotides. For example,GDF-8 polynucleotide may be subjected to site-directed mutagenesis. Thepolynucleotide sequence for GDF-8 also includes antisense sequences. Thepolynucleotides of the invention include sequences that are degenerateas a result of the genetic code. There are 20 natural amino acids, mostof which are specified by more than one codon. Therefore, all degeneratenucleotide sequences are included in the invention as long as the aminoacid sequence of GDF-8 polypeptide encoded by the nucleotide sequence isfunctionally unchanged.

Specifically disclosed herein is a genomic DNA sequence containing aportion of the GDF-8 gene. The sequence contains an open reading framecorresponding to the predicted C-terminal region of the GDF-8 precursorprotein. The encoded polypeptide is predicted to contain two potentialproteolytic processing sites (KR and RR). Cleavage of the precursor atthe downstream site would generate a mature biologically activeC-terminal fragment of 109 amino acids with a predicted molecular weightof approximately 12,400. Also, disclosed are full length murine (SEQ IDNO: 11) and human (SEQ ID NO: 13) GDF-8 cDNA sequences The murinepre-pro-GDF-8 protein (SEQ ID NO: 12) is 376 amino acids in length,which is encoded by a 2676 base pair nucleotide sequence, beginning atnucleotide 104 and extending to a TGA stop codon at nucleotide 1232. Thehuman GDF-8 protein (SEQ ID NO: 14) is 375 amino acids and is encoded bya 2743 base pair sequence, with the open reading frame beginning atnucleotide 59 and extending to nucleotide 1184.

The C-terminal region to GDF-8 following the putative proteolyticprocessing site shows significant homology to the known members of theTGF-β superfamily. The GDF-8 sequence contains most of the residues thatare highly conserved in other family members (see FIG. 3). Like theTGF-βs and inhibin βs, GDF-8 contains an extra pair of cysteine residuesin addition to the 7 cysteines found in virtually all other familymembers. Among the known family members, GDF-8 is most homologous toVgr-1 (45% sequence identity) (see FIG. 4).

Minor modifications of the recombinant GDF-8 primary amino acid sequencemay result in proteins which have substantially equivalent activity ascompared to the GDF-8 polypeptide described herein. Such modificationsmay be deliberate, as by site-directed mutagenesis, or may bespontaneous. All of the polypeptides produced by these modifications areincluded herein as long as the biological activity of GDF-8 stillexists. Further, deletion of one or more amino acids can also result ina modification of the structure of the resultant molecule withoutsignificantly altering its biological activity. This can lead to thedevelopment of a smaller active molecule which would have broaderutility. For example, one can remove amino or carboxy terminal aminoacids which are not required for GDF-8 biological activity.

The nucleotide sequence encoding the GDF-8 polypeptide of the inventionincludes the disclosed sequence and conservative variations thereof. Theterm “conservative variation” as used herein denotes the replacement ofan amino acid residue by another, biologically similar residue. Examplesof conservative variations include the substitution of one hydrophobicresidue such as isoleucine, valine, leucine or methionine for another,or the substitution of one polar residue for another, such as thesubstitution of arginine for lysine, glutamic for aspartic acid, orglutamine for asparagine, and the like. The term “conservativevariation” also includes the use of a substituted amino acid in place ofan unsubstituted parent amino acid provided that antibodies raised tothe substituted polypeptide also immunoreact with the unsubstitutedpolypeptide.

DNA sequences of the invention can be obtained by several methods. Forexample, the DNA can be isolated using hybridization techniques whichare well known in the art. These include, but are not limited to: 1)hybridization of genomic or cDNA libraries with probes to detecthomologous nucleotide sequences, 2) polymerase chain reaction (PCR) ongenomic DNA or cDNA using primers capable of annealing to the DNAsequence of interest, and 3) antibody screening of expression librariesto detect cloned DNA fragments with shared structural features.

Preferably the GDF-8 polynucleotide of the invention is derived from amammalian organism, and most preferably from a mouse, rat, or human.Screening procedures which rely on nucleic acid hybridization make itpossible to isolate any gene sequence from any organism, provided theappropriate probe is available. Oligonucleotide probes, which correspondto a part of the sequence encoding the protein in question, can besynthesized chemically. This requires that short, oligopeptide stretchesof amino acid sequence must be known. The DNA sequence encoding theprotein can be deduced from the genetic code, however, the degeneracy ofthe code must be taken into account. It is possible to perform a mixedaddition reaction when the sequence is degenerate. This includes aheterogeneous mixture of denatured double-stranded DNA. For suchscreening, hybridization is preferably performed on eithersingle-stranded DNA or denatured double-stranded DNA. Hybridization isparticularly useful in the detection of cDNA clones derived from sourceswhere an extremely low amount of mRNA sequences relating to thepolypeptide of interest are present. In other words, by using stringenthybridization conditions directed to avoid non-specific binding, it ispossible, for example, to allow the autoradiographic visualization of aspecific cDNA clone by the hybridization of the target DNA to thatsingle probe in the mixture which is its complete complement (Wallace,et al., Nucl. Acid Res., 9:879, (1981).

The development of specific DNA sequences encoding GDF-8 can also beobtained by: 1) isolation of double-stranded DNA sequences from thegenomic DNA; 2) chemical manufacture of a DNA sequence to provide thenecessary codons for the polypeptide of interest; and 3) in vitrosynthesis of a double-stranded DNA sequence by reverse transcription ofmRNA isolated from a eukaryotic donor cell. In the latter case, adouble-stranded DNA complement of mRNA is eventually formed which isgenerally referred to as cDNA.

Of the three above-noted methods for developing specific DNA sequencesfor use in recombinant procedures, the isolation of genomic DNA isolatesis the least common. This is especially true when it is desirable toobtain the microbial expression of mammalian polypeptides due to thepresence of introns.

The synthesis of DNA sequences is frequently the method of choice whenthe entire sequence of amino acid residues of the desired polypeptideproduct is known. When the entire sequence of amino acid residues of thedesired polypeptide is not known, the direct synthesis of DNA sequencesis not possible and the method of choice is the synthesis of cDNAsequences. Among the standard procedures for isolating cDNA sequences ofinterest is the formation of plasmid- or phage-carrying cDNA librarieswhich are derived from reverse transcription of mRNA which is abundantin donor cells that have a high level of genetic expression. When usedin combination with polymerase chain reaction technology, even rareexpression products can be cloned. In those cases where significantportions of the amino acid sequence of the polypeptide are known, theproduction of labeled single or double-stranded DNA or RNA probesequences duplicating a sequence putatively present in the target cDNAmay be employed in DNA/DNA hybridization procedures which are carriedout on cloned copies of the CDNA which have been denatured into asingle-stranded form (Jay, et al., Nucl. Acid Res., 11-:2325, 1983).

A CDNA expression library, such as lambda gt11, can be screenedindirectly for GDF-8 peptides having at least one epitope, usingantibodies specific for GDF-8. Such antibodies can be eitherpolyclonally or monoclonally derived and used to detect expressionproduct indicative of the presence of GDF-8 cDNA.

DNA sequences encoding GDF-8 can be expressed in vitro by DNA transferinto a suitable host cell. “Host cells” are cells in which a vector canbe propagated and its DNA expressed. The term also includes any progenyof the subject host cell. It is understood that all progeny may not beidentical to the parental cell since there may be mutations that occurduring replication. However, such progeny are included when the term“host cell” is used.

Methods of stable transfer, meaning that the foreign DNA is continuouslymaintained in the host, are known in the art.

In the present invention, the GDF-8 polynucleotide sequences may beinserted into a recombinant expression vector. The term “recombinantexpression vector” refers to a plasmid, virus or other vehicle known inthe art that has been manipulated by insertion or incorporation of theGDF-8 genetic sequences. Such expression vectors contain a promotersequence which facilitates the efficient transcription of the insertedgenetic sequene of the host. The expression vector typically contains anorigin of replication, a promoter, as well as specific genes which allowphenotypic selection of the transformed cells. Vectors suitable for usein the present invention include, but are not limited to the T7-basedexpression vector for expression in bacteria (Rosenburg, et al., Gene,56:125, 1987), the pMSXND expression vector for expression in mammaliancells (Lee and Nathans, J. Biol. Chem., 263:3521, 1988) andbaculovirus-derived vectors for expression in insect cells. The DNAsegment can be present in the vector operably linked to regluatoryelements, for example, a promoter (e.g., T7, metallothioenin I, orpolyhedrin promoters).

Polynucleotide sequences encoding GDF-8 can be expressed in eitherprokaryotes or eukaryotes. Hosts can include microbial, yeast, insectand mammalian organisms. Methods of expressing DNA sequences havingeukaryotic or viral sequences in prokaryotes are well known in the art.Biologically functional viral and plasmid DNA vectors capable ofexpression and replication in a host are known in the art. Such vectorsare used to incorporate DNA sequences of the invention. Preferably, themature C-terminal region of GDF-8 is expressed from a cDNA clonecontaining the entire coding sequence of GDF-8. Alternatively, theC-terminal portion of GDF-8 can be expressed as a fusion protein withthe pro- region of another member of the TGF-β family or co-expressedwith another pro- region (see for example, Hammonds, et al., Mole.Endocrin. 5:149, 1991; Gray, A., and Mason, A., Science, 247:1328,1990).

Transformation of a host cell with recombinant DNA may be carried out byconventional techniques as are well known to those skilled in the art.Where the host is prokaryotic, such as E. coli, competent cells whichare capable of DNA uptake can be prepared from cells harvested afterexponential growth phase and subsequently treated by the CaCl₂ methodusing procedures well known in the art. Alternatively, MgCl₂or RbCl canbe used. Transformation can also be performed after forming a protoplastof the host cell if desired.

When the host is a eukaryote, such methods of transfection of DNA ascalcium phosphate co-precipitates, conventional mechanical proceduressuch as microinjection, electroporation, insertion of a plasmid encasedin liposomes, or virus vectors may be used. Eukaryotic cells can also becotransformed with DNA sequences encoding the GDF-8 of the invention,and a second foreign DNA molecule encoding a selectable phenotype, suchas the herpes simplex thymidine kinase gene. Another method is to use aeukaryotic viral vector, such as simian virus 40 (SV40) or bovinepapilloma virus, to transiently infect or transform eukaryotic cells andexpress the protein. (see for example, Eukaryotic Viral Vectors, ColdSpring Harbor Laboratory, Gluzman ed., 1982).

Isolation and purification of microbial expressed polypeptide, orfragments thereof, provided by the invention, may be carried out byconventional means including preparative chromatography andimmunological separations involving monoclonal or polyclonal antibodies.

The invention includes antibodies immunoreactive with GDF-8 polypeptideor functional fragments thereof. Antibody which consists essentially ofpooled monoclonal antibodies with differer epitopic specificities, aswell as distinct monoclonal antibody preparations are provided.Monoclonal antibodies are made from antigen containing fragments of theprotein by methods well known to those skilled in the art (Kohler, etal., Nature, 256:495, 1975). The term antibody as used in this inventionis meant to include intact molecules as well as fragments thereof, suchas Fab and F(ab′)₂, which are capable of binding an epitopic determinanton GDF-8.

The term “cell-proliferative disorder” denotes malignant as well asnon-malignant cell populations which often appear to differ from thesurrounding tissue both morphologically and genotypically. Malignantcells (i.e. cancer) develop as a result of a multistep process. TheGDF-8 polynucleotide that is an antisense molecule is useful in treatingmalignancies of the various organ systems, particularly, for example,cells in muscle or adipose tissue. Essentially, any disorder which isetiologically linked to altered expression of GDF-8 could be consideredsusceptible to treatment with a GDF-8 suppressing reagent. One suchdisorder is a malignant cell proliferative disorder, for example.

The invention provides a method for detecting a cell proliferativedisorder of muscle or adipose tissue which comprises contacting ananti-GDF-8 antibody with a cell suspected of having a GDF-8 associateddisorder and detecting binding to the antibody. The antibody reactivewith GDF-8 is labeled with a compound which allows detection of bindingto GDF-8. For purposes of the invention, an antibody specific for GDF-8polypeptide may be used to detect the level of GDF-8 in biologicalfluids and tissues. Any specimen containing a detectable amount ofantigen can be used. A preferred sample of this invention is muscletissue. The level of GDF-8 in the suspect cell can be compared with thelevel in a normal cell to determine whether the subject has aGDF-8-associated cell proliferative disorder. Preferably the subject ishuman.

The antibodies of the invention can be used in any subject in which itis desirable to administer in vitro or in vivo immunodiagnosis orimmunotherapy. The antibodies of the invention are suited for use, forexample, in immunoassays in which they can be utilized in liquid phaseor bound to a solid phase carrier. In addition, the antibodies in theseimmunoassays can be detectably labeled in various ways. Examples oftypes of immunoassays which can utilize antibodies of the invention arecompetitive and non-competitive immunoassays in either a direct orindirect format. Examples of such immunoassays are the radioimmunoassay(RIA) and the sandwich (immunometric) assay. Detection of the antigensusing the antibodies of the invention can be done utilizing immunoassayswhich are run in either the forward, reverse, or simultaneous modes,including immunohistochemical assays on physiological samples. Those ofskill in the art will know, or can readily discern, other immunoassayformats without undue experimentation.

The antibodies of the invention can be bound to many different carriersand used to detect the presence of an antigen comprising the polypeptideof the invention. Examples of well-known carriers include glass,polystyrene, polypropylene, polyethylene, dextran, nylon, amylases,natural and modified celluloses, polyacrylamides, agaroses andmagnetite. The nature of the carrier can be either soluble or insolublefor purposes of the invention. Those skilled in the art will know ofother suitable carriers for binding antibodies, or will be able toascertain such, using routine experimentation.

There are many different labels and methods of labeling known to thoseof ordinary skill in the art. Examples of the types of labels which canbe used in the present invention include enzymes, radioisotopes,fluorescent compounds, colloidal metals, chemiluminescent compounds,phosphorescent compounds, and bioluminescent compounds. Those ofordinary skill in the art will know of other suitable labels for bindingto the antibody, or will be able to ascertain such, using routineexperimentation.

Another technique which may also result in greater sensitivity consistsof coupling the antibodies to low molecular weight haptens. Thesehaptens can then be specifically detected by means of a second reaction.For example, it is common to use such haptens as biotin, which reactswith avidin, or dinitrophenyl, puridoxal, and fluorescein, which canreact with specific anti-hapten antibodies.

In using the monoclonal antibodies of the invention for the in vivodetection of antigen, the detectably labeled antibody is given a dosewhich is diagnostically effective. The term “diagnostically effective”means that the amount of detectably labeled monoclonal antibody isadministered in sufficient quantity to enable detection of the sitehaving the antigen comprising a polypeptide of the invention for whichthe monoclonal antibodies are specific.

The concentration of detectably labeled monoclonal antibody which isadministered should be sufficient such that the binding to those cellshaving the polypeptide is detectable compared to the background.Further, it is desirable that the detectably labeled monoclonal antibodybe rapidly cleared from the circulatory system in order to give the besttarget-to-background signal ratio.

As a rule, the dosage of detectably labeled monoclonal antibody for invivo diagnosis will vary depending on such factors as age, sex, andextent of disease of the individual. Such dosages may vary, for example,depending on whether multiple injections are given, antigenic burden,and other factors known to those of skill in the art.

For in vivo diagnostic imaging, the type of detection instrumentavailable is a major factor in selecting a given radioisotope. Theradioisotope chosen must have a type of decay which is detectable for agiven type of instrument. Still another important factor in selecting aradioisotope for in vivo diagnosis is that deleterious radiation withrespect to the host is minimized. Ideally, a radio-isotope used for invivo imaging will lack a particle emission, but produce a large numberof photons in the 140-250 keV range, which may readily be detected byconventional gamma cameras.

For in vivo diagnosis radioisotopes may be bound to immunoglobulineither directly or indirectly by using an intermediate functional group.Intermediate functional groups which often are used to bindradioisotopes which exist as metallic ions to immunoglobulins are thebifunctional chelating agents such as diethylenetriaminepentacetic acid(DTPA) and ethylenediaminetetraacetic acid (EDTA) and similar molecules.Typical examples of metallic ions which can be bound to the monoclonalantibodies of the invention are ¹¹¹In,⁹⁷Ru,⁶⁷Ga,⁶⁸Ga, ⁷²As,⁸⁹Zr,and²⁰¹Tl.

The monoclonal antibodies of the invention can also be labeled with aparamagnetic isotope for purposes of in vivo diagnosis, as in magneticresonance imaging (MRI) or electron spin resonance (ESR). In general,any conventional method for visualizing diagnostic imaging can beutilized. Usually gamma and positron emitting radioisotopes are used forcamera imaging and paramagnetic isotopes for MRI. Elements which areparticularly useful in such techniques include ¹⁵⁷Gd,⁵⁵Mn,¹⁶²Dy,⁵²Cr,and⁵⁶Fe.

The monoclonal antibodies of the invention can be used in vitro and invivo to monitor the course of amelioration of a GDF-8-associated diseasein a subject Thus, for example, by measuring the increase or decrease inthe number of cells expressing antigen comprising a polypeptide of theinvention or changes in the concentration of such antigen present invarious body fluids, it would be possible to determine whether aparticular therapeutic regimen aimed at ameliorating theGDF-8-associated disease is effective. The term “ameliorate” denotes alessening of the detrimental effect of the GDF-8-associated disease inthe subject receiving therapy.

The present invention identifies a nucleotide sequence that can beexpressed in an altered manner as compared to expression in a normalcell, therefore it is possible to design appropriate therapeutic ordiagnostic techniques directed to this sequence. Tnus, where acell-proliferative disorder is associated with the expression of GDF-8.nucleic acid sequences that interfere with GDF-8 expression at thetranslational level can be used. This approach utilizes, for example,antisense nucleic acid and ribozymes to block translation of a specificGDF-8 mRNA, either by masking that mRNA with an antisense nucleic acidor by cleaving it with a ribozyme. Such disorders includeneurodegenerative diseases, for example.

Antisense nucleic acids are DNA or RNA molecules that are complementaryto at least a portion of a specific mRNA molecule (Weintraub, ScientificAmerican, 262:40, 1990). In the cell, the antisense nucleic acidshybridize to the corresponding mRNA, forming a double-stranded molecule.The antisense nucleic acids interfere with the translation of the mRNA,since the cell will not translate a mRNA that is double-stranded.Antisense oligomers of about 15 nucleotides are preferred, since theyare easily synthesized and are less likely to cause problems than largermolecules when introduced into the target GDF-8-producing cell. The useof antisense methods to inhibit the in vitro translation of genes iswell known in the art (Marcus-Sakura, Anal.Biochem., 172:289, 1988).

Ribozymes are RNA molecules possessing the ability to specificallycleave other single-stranded RNA in a manner analogous to DNArestriction endonucleases. Through the modification of nucleotidesequences which encode these RNAs, it is possible to engineer moleculesthat recognize specific nucleotide sequences in an RNA molecule andcleave it (Cech, J.Amer.Med. Assn., 260:3030, 1988). A major advantageof this approach is that, because they are sequence-specific, only mRNAswith particular sequences are inactivated.

There are two basic types of ribozymes namely, tetrahymena-type(Hasselhoff, Nature, 334:585, 1988) and “hammerhead”-type.Tetrahymena-type ribozymes recognize sequences which are four bases inlength, while “hammerhead”-type ribozymes recognize base sequences 11-18bases in length. The longer the recognition sequence, the greater thelikelihood that the sequence will occur exclusively in the target mRNAspecies. Consequently, hammerhead-type ribozymes are preferable totetrahymena-type ribozymes for inactivating a specific mRNA species and18-based recognition sequences are preferable to shorter recognitionsequences.

The present invention also provides gene therapy for the treatment ofcell proliferative or immunologic disorders which are mediated by GDF-8protein. Such therapy would achieve its therapeutic effect byintroduction of the GDF-8 antisense polynucleotide into cells having theproliferative disorder. Delivery of antisense GDF-8 polynucleotide canbe achieved using a recombinant expression vector such as a chimericvirus or a colloidal dispersion system. Especially preferred fortherapeutic delivery of antisense sequences is the use of targetedliposomes.

Various viral vectors which can be utilized for gene therapy as taughtherein include adenovirus, herpes virus, vaccinia, or, preferably, anRNA virus such as a retrovirus. Preferably, the retroviral vector is aderivative of a murine or avian retrovirus. Examples of retroviralvectors in which a single foreign gene can be inserted include, but arenot limited to: Moloney murine leukemia virus (MoMuLV), Harvey murinesarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), and RousSarcoma Virus (RSV). A number of additional retroviral vectors canincorporate multiple genes. All of these vectors can transfer orincorporate a gene for a selectable marker so that transduced cells canbe identified and generated. By inserting a GDF-8 sequence of interestinto the viral vector, along with another gene which encodes the ligandfor a receptor on a specific target cell, for example, the vector is nowtarget specific. Retroviral vectors can be made target specific byattaching, for example, a sugar, a glycolipid, or a protein. Preferredtargeting is accomplished by using an antibody to target the retroviralvector. Those of skill in the art will know of, or can readily ascertainwithout undue experimentation, specific polynucleotide sequences whichcan be inserted into the retroviral genome or attached to a viralenvelope to allow target specific delivery of the retroviral vectorcontaining the GDF-8 antisense polynucleotide.

Since recombinant retroviruses are defective, they require assistance inorder to produce infectious vector particles. This assistance can beprovided, for example, by using helper cell lines that contain plasmidsencoding all of the structural genes of the retrovirus under the controlof regulatory sequences within the LTR. These plasmids are missing anucleotide sequence which enables the packaging mechanism to recognizean RNA transcript for encapsidation. Helper cell lines which havedeletions of the packaging signal include, but are not limited to ψ2,PA317 and PA12, for example. These cell lines produce empty virions,since no genome is packaged. If a retroviral vector is introduced intosuch cells in which the packaging signal is intact, but the structuralgenes are replaced by other genes of interest, the vector can bepackaged and vector virion produced.

Alternatively, NIH 3T3 or other tissue culture cells can be directlytransfected with plasmids encoding the retroviral structural genes gag,pal and env, by conventional calcium phosphate transfection. These cellsare then transfected with the vector plasmid containing the genes ofinterest. The resulting cells release the retroviral vector into theculture medium.

Another targeted delivery system for GDF-8 antisense polynucleotides isa colloidal dispersion system. Colloidal dispersion systems includemacromolecule complexes, nanocapsules, microspheres, beads, andlipid-based systems including oil-in-water emulsions, micelles, mixedmicelles, and liposomes. The preferred colloidal system of thisinvention is a liposome. Liposomes are artificial membrane vesicleswhich are useful as delivery vehicles in vitro and in vivo. It has beenshown that large unilamellar vesicles (LUV), which range in size from0.2-4.0 μm can encapsulate a substantial percentage of an aqueous buffercontaining large macromolecules. RNA, DNA and intact virions can beencapsulated within the aqueous interior and be delivered to cells in abiologically active form (Fraley, et al., Trends Biochem. Sci., 6:77,1981). In addition to mammalian cells, liposomes have been used fordelivery of polynucleotides in plant, yeast and bacterial cells. Inorder for a liposome to be an efficient gene transfer vehicle, thefollowing characteristics should be present: (1) encapsulation of thegenes of interest at high efficiency while not compromising theirbiological activity; (2) preferential and substantial binding to atarget cell in comparison to non-target cells; (3) delivery of theaqueous contents of the vesicle to the target cell cytoplasm at highefficiency; and (4) accurate and effective expression of geneticinformation (Mannino, et al., Biotechniques, 6:682, 1988).

The composition of the liposome is usually a combination ofphospholipids, particularly high-phase-transition-temperaturephospholipids, usually in combination with steroics, especiallycholesterol. Other phospholipids or other lipids may also be used. Thephysical characteristics of liposomes depend on pH, ionic strength, andthe presence of divalent cations.

Examples of lipids useful in liposome production include phosphatidylcompounds, such as phosphatidylglycerol, phosphatidylcholine,phosphatidylserine, phosphatidylethanolamine, sphingolipids,cerebrosides, and gangliosides. Particularly useful arediacylphosphatidylglycerols, where the lipid moiety contains from 14-18carbon atoms, particularly from 16-18 carbon atoms, and is saturated.Illustrative phospholipids include egg phosphatidylcholine,dipalmitoylphosphatidylcholine and distearoylphosphatidylcholine.

The targeting of liposomes can be classified based on anatomical andmechanistic factors. Anatomical classification is based on the level ofselectivity, for example, organ-specific, cell-specific, andorganelle-specific. Mechanistic targeting can be distinguished basedupon whether it is passive or active. Passive targeting utilizes thenatural tendency of liposomes to distribute to cells of thereticulo-endothelial system (RES) in organs which contain sinusoidalcapillaries. Active targeting, on the other hand, involves alteration ofthe liposome by coupling the liposome to a specific ligand such as amonoclonal antibody, sugar, glycolipid, or protein, or by changing thecomposition or size of the liposome in order to achieve targeting toorgans and cell types other than the naturally occurring sites oflocalization.

The surface of the targeted delivery system may be modified in a varietyof ways. In the case of a liposomal targeted delivery system, lipidgroups can be incorporated into the lipid bilayer of the liposome inorder to maintain the targeting ligand in stable association with theliposomal bilayer. Various linking groups can be used for joining thelipid chains to the targeting ligand.

Due to the expression of GDF-8 in muscle and adipose tissue, there are avariety of applications using the polypeptide, polynucleotide, andantibodies of the invention, related to these tissues. Such applicationsinclude treatment of cell proliferative disorders involving these andother tissues, such as neural tissue. In addition, GDF-8 may be usefulin various gene therapy procedures.

The data in Example 6 shows that the human GDF-8 gene is located onchromosome 2. By comparing the chromosomal location of GDF-8 with themap positions of various human disorders, it should be possible todetermine whether mutations in the GDF-8 gene are involved in theetiology of human diseases. For example, an autosomal recessive form ofjuvenile amyotrophic lateral sclerosis has been shown to map tochromosome 2 (Hentati, et al., Neurology, 42 [Suppl.3]:201, 1992). Moreprecise mapping of GDF-8 and analysis of DNA from these patients mayindicate that GDF-8 is, in fact, the gene affected in this disease. Inaddition, GDF-8 is useful for distinguishing chromosome 2 from otherchromosomes.

The following examples are intended to illustrate but not limit theinvention. While they are typical of those that might be used, otherprocedures known to those skilled in the art may alternatively be used.

EXAMPLE 1

Identification and Isolation of a Novel TGF-β Family Member

To identify a new member of the TGF-β superfamily, degenerateoligonucleotides were designed which corresponded to two conservedregions among the known family members: one region spanning the twotryptophan residues conserved in all family members except MIS and theother region spanning the invariant cysteine residues near theC-terminus. These primers were used for polymerase chain reactions onmouse genomic DNA followed by subcloning the PCR products usingrestriction sites placed at the 5′ ends of the primers, pickingindividual E. coli colonies carrying these subcloned inserts, and usinga combination of random sequencing and hybridization analysis toeliminate known members of the superfamily.

GDF-8 was identified from a mixture of PCR products obtained with theprimers

SJL141:5′-CCGGAATTCGGITGG(G/C/A)A(G/A/T/C)(A/G)A(T/C)TGG(A/G)TI(A/G)TI(T/G)CICC-3′  (SEQID NO:1)

SJL147:5′-CCGGAATTC(G/A)CAI(G/C)C(G/A)CA(G/A)CT(G/A/T/C)TCIACI(G/A)(T/C)CAT-3′  (SEQID NO:2)

PCR using these primers was carried out with 2 μg mouse genomic DNA at94° C. for 1 min, 50° C. for 2 min, and 72° C. for 2 min for 40 cycles.

PCR products of approximately 280 bp were gel-purified, digested withEco RI, gel-purified again, and subcloned in the Bluescript vector(Stratagene, San Diego, Calif.). Bacterial colonies carrying individualsubclones were picked into 96 well microtiter plates, and multiplereplicas were prepared by plating the cells onto nitrocellulose. Thereplicate filters were hybridized to probes representing known membersof the family, and DNA was prepared from non-hybridizing colonies forsequence analysis.

The primer combination of SJL141 and SJL147, encoding the amino acidsequences GW(H/Q/N/K/D/E)(D/N)W(V/I/M)(V/I/M)(A/S)P (SEQ ID NO:9) andM(V/I/M/T/A)V(D/E)SC(G/A)C (SEQ ID NO:10), respectively, yielded fourpreviously identified sequences (BMP-4, inhibin βB, GDF-3 and GDF-5) andone novel sequence, which was designated GDF-8, among 110 subclonesanalyzed.

Human GDF-8 was isolated using the primers:

ACM13:5′-CGCGGATCCAGAAGTCAAGGTGACAGACACAC-3′  (SEQ ID NO:3);

and

ACM14:5′-CGCGGATCCTCCTCATGAGCACCCACAGCGGTC-3′  (SEQ ID NO:4)

PCR using these primers was carried out with one μg human genomic DNA at94° C. for 1 min, 58° C. for 2 min, and 72° C. for 2 min for 30 cycles.The PCR product was digested with Bam HI, gel-purified, and subcloned inthe Bluescript vector (Stratagene, San Francisco, Calif.).

EXAMPLE 2

Expression Pattern and Sequence of GDF-8

To determine the expression pattern of GDF-8, RNA samples prepared froma variety of adult tissues were screened by Northern analysis. RNAisolation and Northern analysis were carried out as described previously(Lee, S. -J., Mol. Endocrinol., 4:1034, 1990) except that hybridizationwas carried out in 5×SSPE, 10% dextran sulfate, 50% formamide, 1% SDS,200 μg/ml salmon DNA, and 0.1% each of bovine serum albumin, ficoll, andpolyvinylpyrrolidone. Five micrograms of twice poly A-selected RNAprepared from each tissue (except for muscle, for which only 2 μg RNAwas used) were electrophoresed on formaldehyde gels, blotted, and probedwith GDF-8. As shown in FIG. 1, the GDF-8 probe detected a single mRNAspecies expressed at highest levels in muscle and at significantly lowerlevels in adipose tissue.

To obtain a larger segment of the GDF-8 gene, a mouse genomic librarywas screened with a probe derived from the GDF-8 PCR product. Thepartial sequence of a GDF-8 genomic clone is shown in FIG. 2a. Thesequence contains an open reading frame corresponding to the predictedC-terminal region of the GDF-8 precursor protein. The predicted GDF-8sequence contains two potential proteolytic processing sites, which areboxed. Cleavage of the precursor at the second of these sites wouldgenerate a mature C-terminal fragment 109 amino acids in length with apredicted molecular weight of 12,400. The partial sequence of humanGDF-8 is shown in FIG. 2b. Assuming no PCR-induced errors during theisolation of the human clone, the human and mouse amino acid sequencesin this region are 100% identical.

The C-terminal region of GDF-8 following the putative proteolyticprocessing site shows significant homology to the known members of theTGF-β superfamily (FIG. 3). FIG. 3 shows the alignment of the C-terminalsequences of GDF-8 with the corresponding regions of human GDF-1 (Lee,Proc. Natl . Acad. Sci. USA, 88:4250-4254, 1991), human BMP-2 and 4(Wozney, et al., Science, 242:1528-1534, 1988), human Vgr-1 (Celeste, etal., Proc. Natl. Acad. Sci. USA, 87:9843-9847, 1990), human OP-1(Ozkaynak, et al., EMBO J., 9:2085-2093, 1990), human BMP-5 (Celeste, etal., Proc. Natl. Acad. Sci. USA, 87:9843-9847, 1990), human BMP-3(Wozney, et al., Science, 242:1528-1534, 1988), human MIS (Cate, et al.,Cell, 45:685-698, 1986), human inhibin alpha, βA, and βB (Mason, et al.,Biochem, Biophys. Res. Commun., 135:957-964, 1986), human TGF-β1(Derynck, et al., Nature, 316:701-705, 1985), humanTGF-β2 (deMartin, etal., EMBO J., 6.3673-3677, 1987), and human TGF-β3 (ten Dijke, et al.,Proc. Natl. Acad. Sci. USA, 85:4715-4719 1988). The conserved cysteineresidues are boxed. Dashes denote gaps introduced in order to maximizethe alignment.

GDF-8 contains most of the residues that are highly conserved in otherfamily members, including the seven cysteine residues with theircharacteristic spacing. Like the TGF-βs and inhibin βs, GDF-8 alsocontains two additional cysteine residues. In the case of TGF-β2, thesetwo additional cysteine residues are known to form an intramoleculardisulfide bond (Daopin, et al., Science, 257:369, 1992; Schlunegger andGrutter, Nature, 358:430, 1992).

FIG. 4 shows the amino acid homologies among the different members ofthe TGF-β superfamily. Numbers represent percent amino acid identitiesbetween each pair calculated from the first conserved cysteine to theC-terminus. Boxes represent homologies among highly-related memberswithin particular subgroups. In this region, GDF-8 is most homologous toVgr-1 (45% sequence identity).

EXAMPLE 3

Isolation of cDNA Clones Encoding Murine and Human GDF-8

In order to isolate full-length cDNA clones encoding murine and humanGDF-8, cDNA libraries were prepared in the lambda ZAP II vector(Stratagene) using RNA prepared from skeletal muscle. From 5 μg of twicepoly A-selected RNA prepared from murine and human muscle, cDNAlibraries consisting of 4.4 million and 1.9 million recombinant phage,respectively, were constructed according to the instructions provided byStratagene. These libraries were screened without amplification. Libraryscreening and characterization of cDNA inserts were carried out asdescribed previously (Lee, Mol. Endocrinol, 4:1034-1040).

From 2.4×10⁶ recombinant phage screened from the murine muscle CDNAlibrary, greater than 280 positive phage were identified using a murineGDF-8 probe derived from a genomic clone, as described in Example 1. Theentire nucleotide sequence of the longest cDNA insert analyzed is shownin FIGS. 5a and 5 b. The 2676 base pair sequence contains a single longopen reading frame beginning with a methionine codon at nucleotide 104and extending to a TGA stop codon at nucleotide 1232. Upstream of theputative initiating methionine codon is an in-frame stop codon atnucleotide 23. The predicted pre-pro-GDF-8 protein is 376 amino acids inlength. The sequence contains a core of hydrophobic amino acids at theN-terminus suggestive of a signal peptide for secretion (FIG. 6a), onepotential N-glycosylation site at asparagine 72, a putative RXXR (SEQ IDNO:32) proteolytic cleavage site at amino acids 264-267, and aC-terminal region showing significant homology to the known members ofthe TGF-β superfamily. Cleavage of the precursor protein at the putativeRXXR site would generate a mature C-terminal GDF-8 fragment 109 aminoacids in length with a predicted molecular weight of approximately12,400.

From 1.9×10⁶ recombinant phage screened from the human muscle cDNAlibrary, 4 positive phage were identified using a human GDF-8 probederived by polymerase chain reaction on human genomic DNA. The entirenucleotide sequence of the longest cDNA insert is shown in FIGS. 5c and5 d SEQ ID NO:13. The 2743 base pair sequence contains a single longopen reading frame beginning with a methionine codon at nucleotide 59and extending to a TGA stop codon at nucleotide 1184. The predictedpre-pro-GDF-8 protein is 375 amino acids in length. The sequencecontains a core of hydrophobic amino acids at the N-terminus suggestiveof a signal peptide for secretion (FIG. 6b), one potentialN-glycosylation site at asparagine 71, and a putative RXXR proteolytic(SEQ ID NO:32) cleavage site at amino acids 236-266. FIG. 7 shows acomparison of the predicted murine (top) and human (bottom) GDF-8 aminoacid sequences. Numbers indicate amino acid position relative to theN-terminus. Identities between the two sequences are denoted by avertical line. Murine and human GDF-8 are approximately 94% identical inthe predicted pro-regions and 100% identical following the predictedRXXR (SEQ ID NO:32) cleavage sites.

EXAMPLE 4

Preparation of Antibodies Against GDF-8 and Expression of GDF-8 inMammalian Cells

In order to prepare antibodies against GDF-8, GDF-8 antigen wasexpressed as a fusion protein in bacteria. A portion of murine GDF-8cDNA spanning amino acids 268-376 (mature region) was inserted into thepRSET vector (Invitrogen) such that the GDF-8 coding sequence was placedin frame with the initiating methionine codon present in the vector; theresulting construct created an open reading frame encoding a fusionprotein with a molecular weight of approximately 16,600. The fusionconstruct was transformed into BL21 (DE3) (pLysS) cells, and expressionof the fusion protein was induced by treatment withisopropylthio-β-galactoside as described (Rosenberg, et al., Gene,56:125-135). The fusion protein was then purified by metal chelatechromatography according to the instructions provided by Invitrogen. ACoomassie blue-stained gel of unpurified and purified fusion proteins isshown in FIG. 8.

The purified fusion protein was used to immunize both rabbits andchickens. Immunization of rabbits was carried out by Spring Valley Labs(Sykesville, Md.), and immunization of chickens was carried out by HRP,Inc. (Denver, Pa.). Western analysis of sera both from immunized rabbitsand from immunized chickens demonstrated the presence of antibodiesdirected against the fusion protein.

To express GDF-8 in mammalian cells, the murine GDF-8 cDNA sequence fromnucleotides 48-1303 was cloned in both orientations downstream of themetallothionein I promoter in the pMSXND expression vector, this vectorcontains processing signals derived from SV40, a dihydrofolate reductasegene, and a gene conferring resistance to the antibiotic G418 (Lee andNathans. J. Biol. Chem., 263:3521-3527). The resulting constructs weretransfected into Chinese hamster ovary cells, and stable tranfectantswere selected in the presence of G418. Two milliliters of conditionedmedia prepared from the G418-resistant cells were dialyzed, lyophilized,electrophoresed under denaturing, reducing conditions, transferred tonitrocellulose, and incubated with anti-GDF-8 antibodies (describedabove) and [¹²⁵l]iodoproteinA.

As shown in FIG. 9, the rabbit GDF-8 antibodies (at a 1:500 dilution)detected a protein of approximately the predicted molecular weight forthe mature C-terminal fragment of GDF-8 in the conditioned media ofcells transfected with a construct in which GDF-8 had been cloned in thecorrect (sense) orientation with respect to the metallothionein promoter(lane 2); this band was not detected in a similar sample prepared fromcells transfected with a control antisense construct (lane 1). Similarresults were obtained using antibodies prepared in chickens. Hence,GDF-8 is secreted and proteolytically processed by these transfectedmammalian cells.

EXAMPLE 5

Expression Pattern of GDF-8

To determine the pattern of GDF-8, 5 μg of twice poly A-selected RNAprepared from a variety of murine tissue sources were subjected toNorthern analysis. As shown in FIG. 10a (and as shown previously inExample 2), the GDF-8 probe detected a single mRNA species presentalmost exclusively in skeletal muscle among a large number of adulttissues surveyed. On longer exposures of the same blot, significantlylower but detectable levels of GDF-8 mRNA were seen in fat, brain,thymus, heart, and lung. Hence, these results confirm the high degree ofspecificity of GDF-8 expression in skeletal muscle. GDF-8 mRNA was alsodetected in mouse embryos at both gestational ages (day 12.5 and day18.5 post-coital) examined but not in placentas at various stages ofdevelopment (FIG. 10b).

EXAMPLE 6

Chromosomal Localization of GDF-8

In order to map the chromosomal location of GDF-8. DNA samples fromhuman/rodent somatic cell hybrids (Drwinga, et al., Genomics,16:311-413, 1993; Dubois and Naylor, Genomics, 16:315-319, 1993) wereanalyzed by polymerase chain reaction followed by Southern blotting.Polymerase chain reaction was carried out using primer #83,5′-CGCGGATCCGTGGATCTAAATGAGAACAGTGAGC-3′ (SEQ ID NO:15) and primer #84,5′-CGCGAATTCTCAGGTAATGATTGTTTCCGTTGTAGCG-3′(SEQ ID NO:16) for 40 cyclesat 94° C. for 2 minutes, 60° C. for 1 minute, and 72° C. for 2 minutes.These primers correspond to nucleotides 119 to 143 (flanked by a Bam H1recognition sequence), and nucleotides 394 to 418 (flanked by an Eco R1recognition sequence), respectively, in the human GDF-8 cDNA sequence.PCR products were electrophoresed on agarose gels, blotted, and probedwith oligonucleotide #100, 5′-ACACTAAATCTTCAAGAATA-3′ (SEQ ID NO:17),which corresponds to a sequence internal to the region flanked by primer#83 and #84. Filters were hybridized in 6×SSC, 1×Denhardt's solution,100 μg/ml yeast transfer RNA, and 0.05% sodium pyrophosphate at 50° C.

As shown in FIG. 11, the human-specific probe detected a band of thepredicted size (approximately 320 base pairs) in the positive controlsample (total human genomic DNA) and in a single DNA sample from thehuman/rodent hybrid panel. This positive signal corresponds to humanchromosome 2. The human chromosome contained in each of the hybrid celllines is identified at the top of each of the first 24 lanes (1-22, X,and Y). In the lanes designated M, CHO, and H, the starting DNA templatewas total genomic DNA from mouse, hamster, and human sources,respectively. In the lane marked B1, no template DNA was used. Numbersat left indicate the mobilities of DNA standards. These data show thatthe human GDF-8 gene is located on chromosome 2.

Although the invention has been described with reference to thepresently preferred embodiment, it should be understood that variousmodifications can be made without departing from the spirit of theinvention. Accordingly, the invention is limited only by the followingclaims.

32 35 base pairs nucleic acid single linear Genomic DNA SJL141 ModifiedBase 1...35 /note= “N=inosine” 1 CCGGAATTCG GNTGGVANRA YTGGRTNRTN KCNCC35 33 base pairs nucleic acid single linear Genomic DNA SJL147 CDS1...33 /note= “N-inosine” 2 CCGGAATTCR CANSCRCARC TNTCNACNRY CAT 33 32base pairs nucleic acid single linear ACM13 CDS 1...32 3 CGCGGATCCAGAAGTCAAGG TGACAGACAC AC 32 33 base pairs nucleic acid single linearGenomic DNA ACM14 CDS 1...33 4 CGCGGATCCT CCTCATGAGC ACCCACAGCG GTC 33550 base pairs nucleic acid single linear mouse GDF-8 CDS 59...436 5TTAAGGTAGG AAGGATTTCA GGCTCTATTT ACATAATTGT TCTTTCCTTT TCACACAG 58 AATCCC TTT TTA GAA GTC AAG GTG ACA GAC ACA CCC AAG AGG TCC CGG 106 Asn ProPhe Leu Glu Val Lys Val Thr Asp Thr Pro Lys Arg Ser Arg 1 5 10 15 AGAGAC TTT GGG CTT GAC TGC GAT GAG CAC TCC ACG GAA TCC CGG TGC 154 Arg AspPhe Gly Leu Asp Cys Asp Glu His Ser Thr Glu Ser Arg Cys 20 25 30 TGC CGCTAC CCC CTC ACG GTC GAT TTT GAA GCC TTT GGA TGG GAC TGG 202 Cys Arg TyrPro Leu Thr Val Asp Phe Glu Ala Phe Gly Trp Asp Trp 35 40 45 ATT ATC GCACCC AAA AGA TAT AAG GCC AAT TAC TGC TCA GGA GAG TGT 250 Ile Ile Ala ProLys Arg Tyr Lys Ala Asn Tyr Cys Ser Gly Glu Cys 50 55 60 GAA TTT GTG TTTTTA CAA AAA TAT CCG CAT ACT CAT CTT GTG CAC CAA 298 Glu Phe Val Phe LeuGln Lys Tyr Pro His Thr His Leu Val His Gln 65 70 75 80 GCA AAC CCC AGAGGC TCA GCA GGC CCT TGC TGC ACT CCG ACA AAA ATG 346 Ala Asn Pro Arg GlySer Ala Gly Pro Cys Cys Thr Pro Thr Lys Met 85 90 95 TCT CCC ATT AAT ATGCTA TAT TTT AAT GGC AAA GAA CAA ATA ATA TAT 394 Ser Pro Ile Asn Met LeuTyr Phe Asn Gly Lys Glu Gln Ile Ile Tyr 100 105 110 GGG AAA ATT CCA GCCATG GTA GTA GAC CGC TGT GGG TGC TCA TGAGCTT 446 Gly Lys Ile Pro Ala MetVal Val Asp Arg Cys Gly Cys Ser 115 120 125 ATTAGGTTAG AAACTTCCCAAGTCATGGAA GGTCTTCCCC TCAATTTCGA AACTGTGA 506 TCCTGCAGCC CGGGGGATCCACTAGTTCTA GAGCGGCCGC CACC 550 126 amino acids amino acid linear proteininternal 6 Asn Pro Phe Leu Glu Val Lys Val Thr Asp Thr Pro Lys Arg SerArg 1 5 10 15 Arg Asp Phe Gly Leu Asp Cys Asp Glu His Ser Thr Glu SerArg Cys 20 25 30 Cys Arg Tyr Pro Leu Thr Val Asp Phe Glu Ala Phe Gly TrpAsp Trp 35 40 45 Ile Ile Ala Pro Lys Arg Tyr Lys Ala Asn Tyr Cys Ser GlyGlu Cys 50 55 60 Glu Phe Val Phe Leu Gln Lys Tyr Pro His Thr His Leu ValHis Gln 65 70 75 80 Ala Asn Pro Arg Gly Ser Ala Gly Pro Cys Cys Thr ProThr Lys Met 85 90 95 Ser Pro Ile Asn Met Leu Tyr Phe Asn Gly Lys Glu GlnIle Ile Tyr 100 105 110 Gly Lys Ile Pro Ala Met Val Val Asp Arg Cys GlyCys Ser 115 120 125 326 base pairs nucleic acid single linear humanGDF-8 CDS 3...326 7 CA AAA AGA TCC AGA AGG GAT TTT GGT CTT GAC TGT GATGAG CAC TCA 47 Lys Arg Ser Arg Arg Asp Phe Gly Leu Asp Cys Asp Glu HisSer 1 5 10 15 ACA GAA TCA CGA TGC TGT CGT TAC CCT CTA ACT GTG GAT TTTGAA GCT 95 Thr Glu Ser Arg Cys Cys Arg Tyr Pro Leu Thr Val Asp Phe GluAla 20 25 30 TTT GGA TGG GAT TGG ATT ATC GCT CCT AAA AGA TAT AAG GCC AATTAC 143 Phe Gly Trp Asp Trp Ile Ile Ala Pro Lys Arg Tyr Lys Ala Asn Tyr35 40 45 TGC TCT GGA GAG TGT GAA TTT GTA TTT TTA CAA AAA TAT CCT CAT ACT191 Cys Ser Gly Glu Cys Glu Phe Val Phe Leu Gln Lys Tyr Pro His Thr 5055 60 CAT CTG GTA CAC CAA GCA AAC CCC AGA GGT TCA GCA GGC CCT TGC TGT239 His Leu Val His Gln Ala Asn Pro Arg Gly Ser Ala Gly Pro Cys Cys 6570 75 ACT CCC ACA AAG ATG TCT CCA ATT AAT ATG CTA TAT TTT AAT GGC AAA287 Thr Pro Thr Lys Met Ser Pro Ile Asn Met Leu Tyr Phe Asn Gly Lys 8085 90 95 GAA CAA ATA ATA TAT GGG AAA ATT CCA GCG ATG GTA GTA 326 Glu GlnIle Ile Tyr Gly Lys Ile Pro Ala Met Val Val 100 105 108 amino acidsamino acid linear protein internal 8 Lys Arg Ser Arg Arg Asp Phe Gly LeuAsp Cys Asp Glu His Ser Thr 1 5 10 15 Glu Ser Arg Cys Cys Arg Tyr ProLeu Thr Val Asp Phe Glu Ala Phe 20 25 30 Gly Trp Asp Trp Ile Ile Ala ProLys Arg Tyr Lys Ala Asn Tyr Cys 35 40 45 Ser Gly Glu Cys Glu Phe Val PheLeu Gln Lys Tyr Pro His Thr His 50 55 60 Leu Val His Gln Ala Asn Pro ArgGly Ser Ala Gly Pro Cys Cys Thr 65 70 75 80 Pro Thr Lys Met Ser Pro IleAsn Met Leu Tyr Phe Asn Gly Lys Glu 85 90 95 Gln Ile Ile Tyr Gly Lys IlePro Ala Met Val Val 100 105 9 amino acids amino acid linear peptideSJL141 Peptide 1...9 /note= “Xaa at position 3=His, Gln, Asn, Lys, Aspor Glu; Xaa at position 4=Asp or Asn; Xaa at positions 6 and 7=Val, Ileor Met; Ala = Xaa at position 8=Ala or Ser” 9 Gly Trp Xaa Xaa Trp XaaXaa Xaa Pro 1 5 8 amino acids amino acid linear peptide SJL147 Peptide1...8 /note= “Xaa at position 2=Ile, Val, Met, Thr or Ala; Xaa atposition 4=Asp or Glu; Xaa at position 7=Gly or Ala” 10 Met Xaa Val XaaSer Cys Xaa Cys 1 5 2676 base pairs nucleic acid single linear GenomicDNA Murine GDF-8 CDS 104...1231 11 GTCTCTCGGA CGGTACATGC ACTAATATTTCACTTGGCAT TACTCAAAAG CAAAAAGAA 60 AAATAAGAAC AAGGGAAAAA AAAAGATTGTGCTGATTTTT AAA ATG ATG CAA AAA 115 Met Met Gln Lys 1 CTG CAA ATG TAT GTTTAT ATT TAC CTG TTC ATG CTG ATT GCT GCT GGC 163 Leu Gln Met Tyr Val TyrIle Tyr Leu Phe Met Leu Ile Ala Ala Gly 5 10 15 20 CCA GTG GAT CTA AATGAG GGC AGT GAG AGA GAA GAA AAT GTG GAA AAA 211 Pro Val Asp Leu Asn GluGly Ser Glu Arg Glu Glu Asn Val Glu Lys 25 30 35 GAG GGG CTG TGT AAT GCATGT GCG TGG AGA CAA AAC ACG AGG TAC TCC 259 Glu Gly Leu Cys Asn Ala CysAla Trp Arg Gln Asn Thr Arg Tyr Ser 40 45 50 AGA ATA GAA GCC ATA AAA ATTCAA ATC CTC AGT AAG CTG CGC CTG GAA 307 Arg Ile Glu Ala Ile Lys Ile GlnIle Leu Ser Lys Leu Arg Leu Glu 55 60 65 ACA GCT CCT AAC ATC AGC AAA GATGCT ATA AGA CAA CTT CTG CCA AGA 355 Thr Ala Pro Asn Ile Ser Lys Asp AlaIle Arg Gln Leu Leu Pro Arg 70 75 80 GCG CCT CCA CTC CGG GAA CTG ATC GATCAG TAC GAC GTC CAG AGG GAT 403 Ala Pro Pro Leu Arg Glu Leu Ile Asp GlnTyr Asp Val Gln Arg Asp 85 90 95 100 GAC AGC AGT GAT GGC TCT TTG GAA GATGAC GAT TAT CAC GCT ACC ACG 451 Asp Ser Ser Asp Gly Ser Leu Glu Asp AspAsp Tyr His Ala Thr Thr 105 110 115 GAA ACA ATC ATT ACC ATG CCT ACA GAGTCT GAC TTT CTA ATG CAA GCG 499 Glu Thr Ile Ile Thr Met Pro Thr Glu SerAsp Phe Leu Met Gln Ala 120 125 130 GAT GGC AAG CCC AAA TGT TGC TTT TTTAAA TTT AGC TCT AAA ATA CAG 547 Asp Gly Lys Pro Lys Cys Cys Phe Phe LysPhe Ser Ser Lys Ile Gln 135 140 145 TAC AAC AAA GTA GTA AAA GCC CAA CTGTGG ATA TAT CTC AGA CCC GTC 595 Tyr Asn Lys Val Val Lys Ala Gln Leu TrpIle Tyr Leu Arg Pro Val 150 155 160 AAG ACT CCT ACA ACA GTG TTT GTG CAAATC CTG AGA CTC ATC AAA CCC 643 Lys Thr Pro Thr Thr Val Phe Val Gln IleLeu Arg Leu Ile Lys Pro 165 170 175 180 ATG AAA GAC GGT ACA AGG TAT ACTGGA ATC CGA TCT CTG AAA CTT GAC 691 Met Lys Asp Gly Thr Arg Tyr Thr GlyIle Arg Ser Leu Lys Leu Asp 185 190 195 ATG AGC CCA GGC ACT GGT ATT TGGCAG AGT ATT GAT GTG AAG ACA GTG 739 Met Ser Pro Gly Thr Gly Ile Trp GlnSer Ile Asp Val Lys Thr Val 200 205 210 TTG CAA AAT TGG CTC AAA CAG CCTGAA TCC AAC TTA GGC ATT GAA ATC 787 Leu Gln Asn Trp Leu Lys Gln Pro GluSer Asn Leu Gly Ile Glu Ile 215 220 225 AAA GCT TTG GAT GAG AAT GGC CATGAT CTT GCT GTA ACC TTC CCA GGA 835 Lys Ala Leu Asp Glu Asn Gly His AspLeu Ala Val Thr Phe Pro Gly 230 235 240 CCA GGA GAA GAT GGG CTG AAT CCCTTT TTA GAA GTC AAG GTG ACA GAC 883 Pro Gly Glu Asp Gly Leu Asn Pro PheLeu Glu Val Lys Val Thr Asp 245 250 255 260 ACA CCC AAG AGG TCC CGG AGAGAC TTT GGG CTT GAC TGC GAT GAG CAC 931 Thr Pro Lys Arg Ser Arg Arg AspPhe Gly Leu Asp Cys Asp Glu His 265 270 275 TCC ACG GAA TCC CGG TGC TGCCGC TAC CCC CTC ACG GTC GAT TTT GAA 979 Ser Thr Glu Ser Arg Cys Cys ArgTyr Pro Leu Thr Val Asp Phe Glu 280 285 290 GCC TTT GGA TGG GAC TGG ATTATC GCA CCC AAA AGA TAT AAG GCC AAT 1027 Ala Phe Gly Trp Asp Trp Ile IleAla Pro Lys Arg Tyr Lys Ala Asn 295 300 305 TAC TGC TCA GGA GAG TGT GAATTT GTG TTT TTA CAA AAA TAT CCG CAC 1075 Tyr Cys Ser Gly Glu Cys Glu PheVal Phe Leu Gln Lys Tyr Pro His 310 315 320 ACT CAT CTT GTG CAC CAA GCAAAC CCC AGA GGC TCA GCA GGC CCT TGC 1123 Thr His Leu Val His Gln Ala AsnPro Arg Gly Ser Ala Gly Pro Cys 325 330 335 340 TGC ACT CCG ACA AAA ATGTCT CCC ATT AAT ATG CTA TAT TTT AAT GGC 1171 Cys Thr Pro Thr Lys Met SerPro Ile Asn Met Leu Tyr Phe Asn Gly 345 350 355 AAA GAA CAA ATA ATA TATGGG AAA ATT CCA GCC ATG GTA GTA GAC CGC 1219 Lys Glu Gln Ile Ile Tyr GlyLys Ile Pro Ala Met Val Val Asp Arg 360 365 370 TGT GGG TGC TCATGAGCTTTGC ATTAGGTTAG AAACTTCCCA AGTCATGGAA GGTCT 1276 Cys Gly Cys Ser375 TCCCCTCAAT TTCGAAACTG TGAATTCAAG CACCACAGGC TGTAGGCCTT GAGTATGCTC1336 TAGTAACGTA AGCACAAGCT ACAGTGTATG AACTAAAAGA GAGAATAGAT GCAATGGTTG1396 GCATTCAACC ACCAAAATAA ACCATACTAT AGGATGTTGT ATGATTTCCA GAGTTTTTGA1456 AATAGATGGA GATCAAATTA CATTTATGTC CATATATGTA TATTACAACT ACAATCTAGG1516 CAAGGAAGTG AGAGCACATC TTGTGGTCTG CTGAGTTAGG AGGGTATGAT TAAAAGGTAA1576 AGTCTTATTT CCTAACAGTT TCACTTAATA TTTACAGAAG AATCTATATG TAGCCTTTGT1636 AAAGTGTAGG ATTGTTATCA TTTAAAAACA TCATGTACAC TTATATTTGT ATTGTATACT1696 TGGTAAGATA AAATTCCACA AAGTAGGAAT GGGGCCTCAC ATACACATTG CCATTCCTAT1756 TATAATTGGA CAATCCACCA CGGTGCTAAT GCAGTGCTGA ATGGCTCCTA CTGGACCTCT1816 CGATAGAACA CTCTACAAAG TACGAGTCTC TCTCTCCCTT CCAGGTGCAT CTCCACACAC1876 ACAGCACTAA GTGTTCAATG CATTTTCTTT AAGGAAAGAA GAATCTTTTT TTCTAGAGGT1936 CAACTTTCAG TCAACTCTAG CACAGCGGGA GTGACTGCTG CATCTTAAAA GGCAGCCAAA1996 CAGTATTCAT TTTTTAATCT AAATTTCAAA ATCACTGTCT GCCTTTATCA CATGGCAATT2056 TTGTGGTAAA ATAATGGAAA TGACTGGTTC TATCAATATT GTATAAAAGA CTCTGAAACA2116 ATTACATTTA TATAATATGT ATACAATATT GTTTTGTAAA TAAGTGTCTC CTTTTATATT2176 TACTTTGGTA TATTTTTACA CTAATGAAAT TTCAAATCAT TAAAGTACAA AGACATGTCA2236 TGTATCACAA AAAAGGTGAC TGCTTCTATT TCAGAGTGAA TTAGCAGATT CAATAGTGGT2296 CTTAAAACTC TGTATGTTAA GATTAGAAGG TTATATTACA ATCAATTTAT GTATTTTTTA2356 CATTATCAAC TTATGGTTTC ATGGTGGCTG TATCTATGAA TGTGGCTCCC AGTCAAATTT2416 CAATGCCCCA CCATTTTAAA AATTACAAGC ATTACTAAAC ATACCAACAT GTATCTAAAG2476 AAATACAAAT ATGGTATCTC AATAACAGCT ACTTTTTTAT TTTATAATTT GACAATGAAT2536 ACATTTCTTT TATTTACTTC AGTTTTATAA ATTGGAACTT TGTTTATCAA ATGTATTGTA2596 CTCATAGCTA AATGAAATTA TTTCTTACAT AAAAATGTGT AGAAACTATA AATTAAAGTG2656 TTTTCACATT TTTGAAAGGC 2676 376 amino acids amino acid linearprotein internal 12 Met Met Gln Lys Leu Gln Met Tyr Val Tyr Ile Tyr LeuPhe Met Leu 1 5 10 15 Ile Ala Ala Gly Pro Val Asp Leu Asn Glu Gly SerGlu Arg Glu Glu 20 25 30 Asn Val Glu Lys Glu Gly Leu Cys Asn Ala Cys AlaTrp Arg Gln Asn 35 40 45 Thr Arg Tyr Ser Arg Ile Glu Ala Ile Lys Ile GlnIle Leu Ser Lys 50 55 60 Leu Arg Leu Glu Thr Ala Pro Asn Ile Ser Lys AspAla Ile Arg Gln 65 70 75 80 Leu Leu Pro Arg Ala Pro Pro Leu Arg Glu LeuIle Asp Gln Tyr Asp 85 90 95 Val Gln Arg Asp Asp Ser Ser Asp Gly Ser LeuGlu Asp Asp Asp Tyr 100 105 110 His Ala Thr Thr Glu Thr Ile Ile Thr MetPro Thr Glu Ser Asp Phe 115 120 125 Leu Met Gln Ala Asp Gly Lys Pro LysCys Cys Phe Phe Lys Phe Ser 130 135 140 Ser Lys Ile Gln Tyr Asn Lys ValVal Lys Ala Gln Leu Trp Ile Tyr 145 150 155 160 Leu Arg Pro Val Lys ThrPro Thr Thr Val Phe Val Gln Ile Leu Arg 165 170 175 Leu Ile Lys Pro MetLys Asp Gly Thr Arg Tyr Thr Gly Ile Arg Ser 180 185 190 Leu Lys Leu AspMet Ser Pro Gly Thr Gly Ile Trp Gln Ser Ile Asp 195 200 205 Val Lys ThrVal Leu Gln Asn Trp Leu Lys Gln Pro Glu Ser Asn Leu 210 215 220 Gly IleGlu Ile Lys Ala Leu Asp Glu Asn Gly His Asp Leu Ala Val 225 230 235 240Thr Phe Pro Gly Pro Gly Glu Asp Gly Leu Asn Pro Phe Leu Glu Val 245 250255 Lys Val Thr Asp Thr Pro Lys Arg Ser Arg Arg Asp Phe Gly Leu Asp 260265 270 Cys Asp Glu His Ser Thr Glu Ser Arg Cys Cys Arg Tyr Pro Leu Thr275 280 285 Val Asp Phe Glu Ala Phe Gly Trp Asp Trp Ile Ile Ala Pro LysArg 290 295 300 Tyr Lys Ala Asn Tyr Cys Ser Gly Glu Cys Glu Phe Val PheLeu Gln 305 310 315 320 Lys Tyr Pro His Thr His Leu Val His Gln Ala AsnPro Arg Gly Ser 325 330 335 Ala Gly Pro Cys Cys Thr Pro Thr Lys Met SerPro Ile Asn Met Leu 340 345 350 Tyr Phe Asn Gly Lys Glu Gln Ile Ile TyrGly Lys Ile Pro Ala Met 355 360 365 Val Val Asp Arg Cys Gly Cys Ser 370375 2743 base pairs nucleic acid single linear Genomic DNA Human GDF-8CDS 59...1183 13 AAGAAAAGTA AAAGGAAGAA ACAAGAACAA GAAAAAAGAT TATATTGATTTTAAAATC 58 ATG CAA AAA CTG CAA CTC TGT GTT TAT ATT TAC CTG TTT ATG CTGATT 106 Met Gln Lys Leu Gln Leu Cys Val Tyr Ile Tyr Leu Phe Met Leu Ile1 5 10 15 GTT GCT GGT CCA GTG GAT CTA AAT GAG AAC AGT GAG CAA AAA GAAAAT 154 Val Ala Gly Pro Val Asp Leu Asn Glu Asn Ser Glu Gln Lys Glu Asn20 25 30 GTG GAA AAA GAG GGG CTG TGT AAT GCA TGT ACT TGG AGA CAA AAC ACT202 Val Glu Lys Glu Gly Leu Cys Asn Ala Cys Thr Trp Arg Gln Asn Thr 3540 45 AAA TCT TCA AGA ATA GAA GCC ATT AAG ATA CAA ATC CTC AGT AAA CTT250 Lys Ser Ser Arg Ile Glu Ala Ile Lys Ile Gln Ile Leu Ser Lys Leu 5055 60 CGT CTG GAA ACA GCT CCT AAC ATC AGC AAA GAT GTT ATA AGA CAA CTT298 Arg Leu Glu Thr Ala Pro Asn Ile Ser Lys Asp Val Ile Arg Gln Leu 6570 75 80 TTA CCC AAA GCT CCT CCA CTC CGG GAA CTG ATT GAT CAG TAT GAT GTC346 Leu Pro Lys Ala Pro Pro Leu Arg Glu Leu Ile Asp Gln Tyr Asp Val 8590 95 CAG AGG GAT GAC AGC AGC GAT GGC TCT TTG GAA GAT GAC GAT TAT CAC394 Gln Arg Asp Asp Ser Ser Asp Gly Ser Leu Glu Asp Asp Asp Tyr His 100105 110 GCT ACA ACG GAA ACA ATC ATT ACC ATG CCT ACA GAG TCT GAT TTT CTA442 Ala Thr Thr Glu Thr Ile Ile Thr Met Pro Thr Glu Ser Asp Phe Leu 115120 125 ATG CAA GTG GAT GGA AAA CCC AAA TGT TGC TTC TTT AAA TTT AGC TCT490 Met Gln Val Asp Gly Lys Pro Lys Cys Cys Phe Phe Lys Phe Ser Ser 130135 140 AAA ATA CAA TAC AAT AAA GTA GTA AAG GCC CAA CTA TGG ATA TAT TTG538 Lys Ile Gln Tyr Asn Lys Val Val Lys Ala Gln Leu Trp Ile Tyr Leu 145150 155 160 AGA CCC GTC GAG ACT CCT ACA ACA GTG TTT GTG CAA ATC CTG AGACTC 586 Arg Pro Val Glu Thr Pro Thr Thr Val Phe Val Gln Ile Leu Arg Leu165 170 175 ATC AAA CCT ATG AAA GAC GGT ACA AGG TAT ACT GGA ATC CGA TCTCTG 634 Ile Lys Pro Met Lys Asp Gly Thr Arg Tyr Thr Gly Ile Arg Ser Leu180 185 190 AAA CTT GAC ATG AAC CCA GGC ACT GGT ATT TGG CAG AGC ATT GATGTG 682 Lys Leu Asp Met Asn Pro Gly Thr Gly Ile Trp Gln Ser Ile Asp Val195 200 205 AAG ACA GTG TTG CAA AAT TGG CTC AAA CAA CCT GAA TCC AAC TTAGGC 730 Lys Thr Val Leu Gln Asn Trp Leu Lys Gln Pro Glu Ser Asn Leu Gly210 215 220 ATT GAA ATA AAA GCT TTA GAT GAG AAT GGT CAT GAT CTT GCT GTAACC 778 Ile Glu Ile Lys Ala Leu Asp Glu Asn Gly His Asp Leu Ala Val Thr225 230 235 240 TTC CCA GGA CCA GGA GAA GAT GGG CTG AAT CCG TTT TTA GAGGTC AAG 826 Phe Pro Gly Pro Gly Glu Asp Gly Leu Asn Pro Phe Leu Glu ValLys 245 250 255 GTA ACA GAC ACA CCA AAA AGA TCC AGA AGG GAT TTT GGT CTTGAC TGT 874 Val Thr Asp Thr Pro Lys Arg Ser Arg Arg Asp Phe Gly Leu AspCys 260 265 270 GAT GAG CAC TCA ACA GAA TCA CGA TGC TGT CGT TAC CCT CTAACT GTG 922 Asp Glu His Ser Thr Glu Ser Arg Cys Cys Arg Tyr Pro Leu ThrVal 275 280 285 GAT TTT GAA GCT TTT GGA TGG GAT TGG ATT ATC GCT CCT AAAAGA TAT 970 Asp Phe Glu Ala Phe Gly Trp Asp Trp Ile Ile Ala Pro Lys ArgTyr 290 295 300 AAG GCC AAT TAC TGC TCT GGA GAG TGT GAA TTT GTA TTT TTACAA AAG 1018 Lys Ala Asn Tyr Cys Ser Gly Glu Cys Glu Phe Val Phe Leu GlnLys 305 310 315 320 TAT CCT CAT ACT CAT CTG GTA CAC CAA GCA AAC CCC AGAGGT TCA GCA 1066 Tyr Pro His Thr His Leu Val His Gln Ala Asn Pro Arg GlySer Ala 325 330 335 GGC CCT TGC TGT ACT CCC ACA AAG ATG TCT CCA ATT AATATG CTA TAT 1114 Gly Pro Cys Cys Thr Pro Thr Lys Met Ser Pro Ile Asn MetLeu Tyr 340 345 350 TTT AAT GGC AAA GAA CAA ATA ATA TAT GGG AAA ATT CCAGCG ATG GTA 1162 Phe Asn Gly Lys Glu Gln Ile Ile Tyr Gly Lys Ile Pro AlaMet Val 355 360 365 GTA GAC CGC TGT GGG TGC TCA TGAGATTTAT ATTAAGCGTTCATAACTTCC TAAAAC 1219 Val Asp Arg Cys Gly Cys Ser 370 375 ATGGAAGGTTTTCCCCTCAA CAATTTTGAA GCTGTGAAAT TAAGTACCAC AGGCTATAGG 1279 CCTAGAGTATGCTACAGTCA CTTAAGCATA AGCTACAGTA TGTAAACTAA AAGGGGGATT 1339 ATATGCAATGGTTGGCATTT AACCATCCAA ACAAATCATA CAAGAAAGTT TTATGATTTC 1399 CAGAGTTTTTGAGCTAGAAG GAGATCAAAT TACATTTATG TTCCTATATA TTACAACATC 1459 GGCGAGGAAATGAAAGCGAT TCTCCTTGAG TTCTGATGAA TTAAAGGAGT ATGCTTTTAA 1519 GTCTATTTCTTTAAAGTTTT GTTTAATATT TACAGAAAAA TCCACATACA GTATTGGTAA 1579 AATGCAGGATTGTTATATAC CATCATTCGA ATCATCCTTA AACACTTGAA TTTATATTGT 1639 ATGGTAGTATACTTGGTAAG ATAAAATTCC ACAAAAATAG GGATGGTGCA GCATATGCAA 1699 TTTCCATTCCTATTATAATT GACACAGTAC ATTAACAATC CATGCCAACG GTGCTAATAC 1759 GATAGGCTGAATGTCTGAGG CTACCAGGTT TATCACATAA AAAACATTCA GTAAAATAGT 1819 AAGTTTCTCTTTTCTTCAGG TGCATTTTCC TACACCTCCA AATGAGGAAT GGATTTTAGT 1879 TAATGTAAGAAGAATCATTT TTCTAGAGGT TGGCTTTCAA TTCTGTAGCA TACTTGGAGA 1939 AACTGCATTATCTTAAAAGG CAGTCAAATG GTGTTTGTTT TTATCAAAAT GTCAAAATAA 1999 CATACTTGGAGAAGTATGTA ATTTTGTCTT TGGAAAATTA CAACACTGCC TTTGCAACAC 2059 TGCAGTTTTTATGGTAAAAT AATAGAAATG ATCGACTCTA TCAATATTGT ATAAAAAGAC 2119 TGAAACAATGCATTTATATA ATATGTATAC AATATTGTTT TGTAAATAAG TGTCTCCTTT 2179 TTTATTTACTTTGGTATATT TTTACACTAA GGACATTTCA AATTAAGTAC TAAGGCACAA 2239 AGACATGTCATGCATCACAG AAAAGCAACT ACTTATATTT CAGAGCAAAT TAGCAGATTA 2299 AATAGTGGTCTTAAAACTCC ATATGTTAAT GATTAGATGG TTATATTACA ATCATTTTAT 2359 ATTTTTTTACATGATTAACA TTCACTTATG GATTCATGAT GGCTGTATAA AGTGAATTTG 2419 AAATTTCAATGGTTTACTGT CATTGTGTTT AAATCTCAAC GTTCCATTAT TTTAATACTT 2479 GCAAAAACATTACTAAGTAT ACCAAAATAA TTGACTCTAT TATCTGAAAT GAAGAATAAA 2539 CTGATGCTATCTCAACAATA ACTGTTACTT TTATTTTATA ATTTGATAAT GAATATATTT 2599 CTGCATTTATTTACTTCTGT TTTGTAAATT GGGATTTTGT TAATCAAATT TATTGTACTA 2659 TGACTAAATGAAATTATTTC TTACATCTAA TTTGTAGAAA CAGTATAAGT TATATTAAAG 2719 TGTTTTCACATTTTTTTGAA AGAC 2743 375 amino acids amino acid linear protein internal14 Met Gln Lys Leu Gln Leu Cys Val Tyr Ile Tyr Leu Phe Met Leu Ile 1 510 15 Val Ala Gly Pro Val Asp Leu Asn Glu Asn Ser Glu Gln Lys Glu Asn 2025 30 Val Glu Lys Glu Gly Leu Cys Asn Ala Cys Thr Trp Arg Gln Asn Thr 3540 45 Lys Ser Ser Arg Ile Glu Ala Ile Lys Ile Gln Ile Leu Ser Lys Leu 5055 60 Arg Leu Glu Thr Ala Pro Asn Ile Ser Lys Asp Val Ile Arg Gln Leu 6570 75 80 Leu Pro Lys Ala Pro Pro Leu Arg Glu Leu Ile Asp Gln Tyr Asp Val85 90 95 Gln Arg Asp Asp Ser Ser Asp Gly Ser Leu Glu Asp Asp Asp Tyr His100 105 110 Ala Thr Thr Glu Thr Ile Ile Thr Met Pro Thr Glu Ser Asp PheLeu 115 120 125 Met Gln Val Asp Gly Lys Pro Lys Cys Cys Phe Phe Lys PheSer Ser 130 135 140 Lys Ile Gln Tyr Asn Lys Val Val Lys Ala Gln Leu TrpIle Tyr Leu 145 150 155 160 Arg Pro Val Glu Thr Pro Thr Thr Val Phe ValGln Ile Leu Arg Leu 165 170 175 Ile Lys Pro Met Lys Asp Gly Thr Arg TyrThr Gly Ile Arg Ser Leu 180 185 190 Lys Leu Asp Met Asn Pro Gly Thr GlyIle Trp Gln Ser Ile Asp Val 195 200 205 Lys Thr Val Leu Gln Asn Trp LeuLys Gln Pro Glu Ser Asn Leu Gly 210 215 220 Ile Glu Ile Lys Ala Leu AspGlu Asn Gly His Asp Leu Ala Val Thr 225 230 235 240 Phe Pro Gly Pro GlyGlu Asp Gly Leu Asn Pro Phe Leu Glu Val Lys 245 250 255 Val Thr Asp ThrPro Lys Arg Ser Arg Arg Asp Phe Gly Leu Asp Cys 260 265 270 Asp Glu HisSer Thr Glu Ser Arg Cys Cys Arg Tyr Pro Leu Thr Val 275 280 285 Asp PheGlu Ala Phe Gly Trp Asp Trp Ile Ile Ala Pro Lys Arg Tyr 290 295 300 LysAla Asn Tyr Cys Ser Gly Glu Cys Glu Phe Val Phe Leu Gln Lys 305 310 315320 Tyr Pro His Thr His Leu Val His Gln Ala Asn Pro Arg Gly Ser Ala 325330 335 Gly Pro Cys Cys Thr Pro Thr Lys Met Ser Pro Ile Asn Met Leu Tyr340 345 350 Phe Asn Gly Lys Glu Gln Ile Ile Tyr Gly Lys Ile Pro Ala MetVal 355 360 365 Val Asp Arg Cys Gly Cys Ser 370 375 34 base pairsnucleic acid single linear Genomic DNA #83 CDS 1..34 15 CGCGGATCCGTGGATCTAAA TGAGAACAGT GAGC 34 37 base pairs nucleic acid single linearGenomic DNA #84 CDS 1..37 16 CGCGAATTCT CAGGTAATGA TTGTTTCCGT TGTAGCG 3720 base pairs nucleic acid single linear Genomic DNA #100 CDS 1..20 17ACACTAAATC TTCAAGAATA 20 123 amino acids amino acid linear protein GDF-1Protein 1..123 18 Arg Pro Arg Arg Asp Ala Glu Pro Val Leu Gly Gly GlyPro Gly Gly 1 5 10 15 Ala Cys Arg Ala Arg Arg Leu Tyr Val Ser Phe ArgGlu Val Gly Trp 20 25 30 His Arg Trp Val Ile Ala Pro Arg Gly Phe Leu AlaAsn Tyr Cys Gln 35 40 45 Gly Gln Cys Ala Leu Pro Val Ala Leu Ser Gly SerGly Gly Pro Pro 50 55 60 Ala Leu Asn His Ala Val Leu Arg Ala Leu Met HisAla Ala Ala Pro 65 70 75 80 Gly Ala Ala Asp Leu Pro Cys Cys Val Pro AlaArg Leu Ser Pro Ile 85 90 95 Ser Val Leu Phe Phe Asp Asn Ser Asp Asn ValVal Leu Arg Gln Tyr 100 105 110 Glu Asp Met Val Val Asp Glu Cys Gly CysArg 115 120 118 amino acids amino acid linear protein BMP-2 Protein1..118 19 Arg Glu Lys Arg Gln Ala Lys His Lys Gln Arg Lys Arg Leu LysSer 1 5 10 15 Ser Cys Lys Arg His Pro Leu Tyr Val Asp Phe Ser Asp ValGly Trp 20 25 30 Asn Asp Trp Ile Val Ala Pro Pro Gly Tyr His Ala Phe TyrCys His 35 40 45 Gly Glu Cys Pro Phe Pro Leu Ala Asp His Leu Asn Ser ThrAsn His 50 55 60 Ala Ile Val Gln Thr Leu Val Asn Ser Val Asn Ser Lys IlePro Lys 65 70 75 80 Ala Cys Cys Val Pro Thr Glu Leu Ser Ala Ile Ser MetLeu Tyr Leu 85 90 95 Asp Glu Asn Glu Lys Val Val Leu Lys Asn Tyr Gln AspMet Val Val 100 105 110 Glu Gly Cys Gly Cys Arg 115 118 amino acidsamino acid linear protein BMP-4 Protein 1..118 20 Lys Arg Ser Pro LysHis His Ser Gln Arg Ala Arg Lys Lys Asn Lys 1 5 10 15 Asn Cys Arg ArgHis Ser Leu Tyr Val Asp Phe Ser Asp Val Gly Trp 20 25 30 Asn Asp Trp IleVal Ala Pro Pro Gly Tyr Gln Ala Phe Tyr Cys His 35 40 45 Gly Asp Cys ProPhe Pro Leu Ala Asp His Leu Asn Ser Thr Asn His 50 55 60 Ala Ile Val GlnThr Leu Val Asn Ser Val Asn Ser Ser Ile Pro Lys 65 70 75 80 Ala Cys CysVal Pro Thr Glu Leu Ser Ala Ile Ser Met Leu Tyr Leu 85 90 95 Asp Glu TyrAsp Lys Val Val Leu Lys Asn Tyr Gln Glu Met Val Val 100 105 110 Glu GlyCys Gly Cys Arg 115 119 amino acids amino acid linear protein Vgr-1Protein 1..119 21 Ser Arg Gly Ser Gly Ser Ser Asp Tyr Asn Gly Ser GluLeu Lys Thr 1 5 10 15 Ala Cys Lys Lys His Glu Leu Tyr Val Ser Phe GlnAsp Leu Gly Trp 20 25 30 Gln Asp Trp Ile Ile Ala Pro Lys Gly Tyr Ala AlaAsn Tyr Cys Asp 35 40 45 Gly Glu Cys Ser Phe Pro Leu Asn Ala His Met AsnAla Thr Asn His 50 55 60 Ala Ile Val Gln Thr Leu Val His Leu Met Asn ProGlu Tyr Val Pro 65 70 75 80 Lys Pro Cys Cys Ala Pro Thr Lys Leu Asn AlaIle Ser Val Leu Tyr 85 90 95 Phe Asp Asp Asn Ser Asn Val Ile Leu Lys LysTyr Arg Asn Met Val 100 105 110 Val Arg Ala Cys Gly Cys His 115 119amino acids amino acid linear protein OP-1 Protein 1..119 22 Leu Arg MetAla Asn Val Ala Glu Asn Ser Ser Ser Asp Gln Arg Gln 1 5 10 15 Ala CysLys Lys His Glu Leu Tyr Val Ser Phe Arg Asp Leu Gly Trp 20 25 30 Gln AspTrp Ile Ile Ala Pro Glu Gly Tyr Ala Ala Tyr Tyr Cys Glu 35 40 45 Gly GluCys Ala Phe Pro Leu Asn Ser Tyr Met Asn Ala Thr Asn His 50 55 60 Ala IleVal Gln Thr Leu Val His Phe Ile Asn Pro Glu Thr Val Pro 65 70 75 80 LysPro Cys Cys Ala Pro Thr Gln Leu Asn Ala Ile Ser Val Leu Tyr 85 90 95 PheAsp Asp Ser Ser Asn Val Ile Leu Lys Lys Tyr Arg Asn Met Val 100 105 110Val Arg Ala Cys Gly Cys His 115 119 amino acids amino acid linearprotein BMP-5 Protein 1..119 23 Ser Arg Met Ser Ser Val Gly Asp Tyr AsnThr Ser Glu Gln Lys Gln 1 5 10 15 Ala Cys Lys Lys His Glu Leu Tyr ValSer Phe Arg Asp Leu Gly Trp 20 25 30 Gln Asp Trp Ile Ile Ala Pro Glu GlyTyr Ala Ala Phe Tyr Cys Asp 35 40 45 Gly Glu Cys Ser Phe Pro Leu Asn AlaHis Met Asn Ala Thr Asn His 50 55 60 Ala Ile Val Gln Thr Leu Val His LeuMet Phe Pro Asp His Val Pro 65 70 75 80 Lys Pro Cys Cys Ala Pro Thr LysLeu Asn Ala Ile Ser Val Leu Tyr 85 90 95 Phe Asp Asp Ser Ser Asn Val IleLeu Lys Lys Tyr Arg Asn Met Val 100 105 110 Val Arg Ser Cys Gly Cys His115 120 amino acids amino acid linear protein BMP-3 Protein 1..120 24Glu Gln Thr Leu Lys Lys Ala Arg Arg Lys Gln Trp Ile Glu Pro Arg 1 5 1015 Asn Cys Ala Arg Arg Tyr Leu Lys Val Asp Phe Ala Asp Ile Gly Trp 20 2530 Ser Glu Trp Ile Ile Ser Pro Lys Ser Phe Asp Ala Tyr Tyr Cys Ser 35 4045 Gly Ala Cys Gln Phe Pro Met Pro Lys Ser Leu Lys Pro Ser Asn His 50 5560 Ala Thr Ile Gln Ser Ile Val Arg Ala Val Gly Val Val Pro Gly Ile 65 7075 80 Pro Glu Pro Cys Cys Val Pro Glu Lys Met Ser Ser Leu Ser Ile Leu 8590 95 Phe Phe Asp Glu Asn Lys Asn Val Val Leu Lys Val Tyr Pro Asn Met100 105 110 Thr Val Glu Ser Cys Ala Cys Arg 115 120 116 amino acidsamino acid linear protein MIS Protein 1..116 25 Gly Pro Gly Arg Ala GlnArg Ser Ala Gly Ala Thr Ala Ala Asp Gly 1 5 10 15 Pro Cys Ala Leu ArgGlu Leu Ser Val Asp Leu Arg Ala Glu Arg Ser 20 25 30 Val Leu Ile Pro GluThr Tyr Gln Ala Asn Asn Cys Gln Gly Val Cys 35 40 45 Gly Trp Pro Gln SerAsp Arg Asn Pro Arg Tyr Gly Asn His Val Val 50 55 60 Leu Leu Leu Lys MetGln Ala Arg Gly Ala Ala Leu Ala Arg Pro Pro 65 70 75 80 Cys Cys Val ProThr Ala Tyr Ala Gly Lys Leu Leu Ile Ser Leu Ser 85 90 95 Glu Glu Arg IleSer Ala His His Val Pro Asn Met Val Ala Thr Glu 100 105 110 Cys Gly CysArg 115 122 amino acids amino acid linear protein Inhibin-alpha Protein1..122 26 Ala Leu Arg Leu Leu Gln Arg Pro Pro Glu Glu Pro Ala Ala HisAla 1 5 10 15 Asn Cys His Arg Val Ala Leu Asn Ile Ser Phe Gln Glu LeuGly Trp 20 25 30 Glu Arg Trp Ile Val Tyr Pro Pro Ser Phe Ile Phe His TyrCys His 35 40 45 Gly Gly Cys Gly Leu His Ile Pro Pro Asn Leu Ser Leu ProVal Pro 50 55 60 Gly Ala Pro Pro Thr Pro Ala Gln Pro Tyr Ser Leu Leu ProGly Ala 65 70 75 80 Gln Pro Cys Cys Ala Ala Leu Pro Gly Thr Met Arg ProLeu His Val 85 90 95 Arg Thr Thr Ser Asp Gly Gly Tyr Ser Phe Lys Tyr GluThr Val Pro 100 105 110 Asn Leu Leu Thr Gln His Cys Ala Cys Ile 115 120122 amino acids amino acid linear protein Inhibin-beta-alpha Protein1..122 27 His Arg Arg Arg Arg Arg Gly Leu Glu Cys Asp Gly Lys Val AsnIle 1 5 10 15 Cys Cys Lys Lys Gln Phe Phe Val Ser Phe Lys Asp Ile GlyTrp Asn 20 25 30 Asp Trp Ile Ile Ala Pro Ser Gly Tyr His Ala Asn Tyr CysGlu Gly 35 40 45 Glu Cys Pro Ser His Ile Ala Gly Thr Ser Gly Ser Ser LeuSer Phe 50 55 60 His Ser Thr Val Ile Asn His Tyr Arg Met Arg Gly His SerPro Phe 65 70 75 80 Ala Asn Leu Lys Ser Cys Cys Val Pro Thr Lys Leu ArgPro Met Ser 85 90 95 Met Leu Tyr Tyr Asp Asp Gly Gln Asn Ile Ile Lys LysAsp Ile Gln 100 105 110 Asn Met Ile Val Glu Glu Cys Gly Cys Ser 115 120121 amino acids amino acid linear protein Inhibin-beta-beta Protein1..121 28 His Arg Ile Arg Lys Arg Gly Leu Glu Cys Asp Gly Arg Thr AsnLeu 1 5 10 15 Cys Cys Arg Gln Gln Phe Phe Ile Asp Phe Arg Leu Ile GlyTrp Asn 20 25 30 Asp Trp Ile Ile Ala Pro Thr Gly Tyr Tyr Gly Asn Tyr CysGlu Gly 35 40 45 Ser Cys Pro Ala Tyr Leu Ala Gly Val Pro Gly Ser Ala SerSer Phe 50 55 60 His Thr Ala Val Val Asn Gln Tyr Arg Met Arg Gly Leu AsnPro Gly 65 70 75 80 Thr Val Asn Ser Cys Cys Ile Pro Thr Lys Leu Ser ThrMet Ser Met 85 90 95 Leu Tyr Phe Asp Asp Glu Tyr Asn Ile Val Lys Arg AspVal Pro Asn 100 105 110 Met Ile Val Glu Glu Cys Gly Cys Ala 115 120 115amino acids amino acid linear protein TGF-beta-1 Protein 1..115 29 HisArg Arg Ala Leu Asp Thr Asn Tyr Cys Phe Ser Ser Thr Glu Lys 1 5 10 15Asn Cys Cys Val Arg Gln Leu Tyr Ile Asp Phe Arg Lys Asp Leu Gly 20 25 30Trp Lys Trp Ile His Glu Pro Lys Gly Tyr His Ala Asn Phe Cys Leu 35 40 45Gly Pro Cys Pro Tyr Ile Trp Ser Leu Asp Thr Gln Tyr Ser Lys Val 50 55 60Leu Ala Leu Tyr Asn Gln His Asn Pro Gly Ala Ser Ala Ala Pro Cys 65 70 7580 Cys Val Pro Gln Ala Leu Glu Pro Leu Pro Ile Val Tyr Tyr Val Gly 85 9095 Arg Lys Pro Lys Val Glu Gln Leu Ser Asn Met Ile Val Arg Ser Cys 100105 110 Lys Cys Ser 115 115 amino acids amino acid linear proteinTGF-beta-2 Protein 1..115 30 Lys Lys Arg Ala Leu Asp Ala Ala Tyr Cys PheArg Asn Val Gln Asp 1 5 10 15 Asn Cys Cys Leu Arg Pro Leu Tyr Ile AspPhe Lys Arg Asp Leu Gly 20 25 30 Trp Lys Trp Ile His Glu Pro Lys Gly TyrAsn Ala Asn Phe Cys Ala 35 40 45 Gly Ala Cys Pro Tyr Leu Trp Ser Ser AspThr Gln His Ser Arg Val 50 55 60 Leu Ser Leu Tyr Asn Thr Ile Asn Pro GluAla Ser Ala Ser Pro Cys 65 70 75 80 Cys Val Ser Gln Asp Leu Glu Pro LeuThr Ile Leu Tyr Tyr Ile Gly 85 90 95 Lys Thr Pro Lys Ile Glu Gln Leu SerAsn Met Ile Val Lys Ser Cys 100 105 110 Lys Cys Ser 115 115 amino acidsamino acid linear protein TGF-beta-3 Protein 1..115 31 Lys Lys Arg AlaLeu Asp Thr Asn Tyr Cys Phe Arg Asn Leu Glu Glu 1 5 10 15 Asn Cys CysVal Arg Pro Leu Tyr Ile Asp Phe Arg Gln Asp Leu Gly 20 25 30 Trp Lys TrpVal His Glu Pro Lys Gly Tyr Tyr Ala Asn Phe Cys Ser 35 40 45 Gly Pro CysPro Tyr Leu Arg Ser Ala Asp Thr Thr His Ser Thr Val 50 55 60 Leu Gly LeuTyr Asn Thr Leu Asn Pro Glu Ala Ser Ala Ser Pro Cys 65 70 75 80 Cys ValPro Gln Asp Leu Glu Pro Leu Thr Ile Leu Tyr Tyr Val Gly 85 90 95 Arg ThrPro Lys Val Glu Gln Leu Ser Asn Met Val Val Lys Ser Cys 100 105 110 LeuCys Ser 115 4 amino acids amino acid linear peptide Peptide 1..118/note= “Xaa at positions 2 and 3 is any amino acid” 32 Arg Xaa Xaa Arg 1

We claim:
 1. An isolated nucleic acid sequence encoding a GrowthDifferentiation Factor-8 (GDF-8) family member polypeptide, said nucleicacid sequence comprising a nucleotide sequence having about 97% homologyto SEQ ID NO:5 or SEQ ID NO:7, or to a nucleotide sequence complementaryto SEQ ID NO:5 or SEQ ID NO:7, wherein said nucleic acid sequenceencodes a polypeptide that regulates muscle cell growth.
 2. A vector,comprising the nucleic acid sequence of claim
 1. 3. The vector of claim2, which is an expression vector.
 4. The vector of claim 3, which is aviral vector.
 5. A host cell, which contains the nucleic acid sequenceof claim
 1. 6. A host cell, which contains the vector of claim
 3. 7. Anisolated nucleic acid sequence encoding a Growth DifferentiationFactor-8 (GDF-8) family member polypeptide, said nucleic acid sequencecomprising a nucleotide sequence having about 90% sequence identity toSEQ ID NO:5 or SEQ ID NO:7 or to a nucleotide sequence complementary toSEQ ID NO:5 or SEQ ID NO:7, wherein said nucleic acid sequence encodes apolypeptide that regulates muscle cell growth.
 8. The isolated nucleicacid sequence of claim 7, wherein said GDF-8 family member polypeptidecomprises an amino acid sequence as set forth in SEQ ID NO:6 or SEQ IDNO:8.
 9. A vector, comprising the nucleic acid sequence of claim
 7. 10.A host cell, which contains the nucleic acid sequence of claim
 7. 11. Anoligonucleotide fragment of the nucleic acid sequence of claim 1,wherein said oligonucleotide is at least 15 nucleotides in length andselectively hybridizes to genomic DNA encoding SEQ ID NO:12 or SEQ IDNO:14.